Combination therapy using a TNF binding protein for treating TNF-mediated diseases

ABSTRACT

The invention relates to methods for treating or preventing acute and/or chronic arthritis. The method comprises administering to patients in need thereof therapeutically effective amounts of a TNF binding protein and methotrexate (N-[4-[[2,4-diamino-6-pteridinyl)methylamino]benzoyl]-L-glutamic acid). In a preferred embodiment, the TNF binding protein is sTNFR-I or sTNFR-II. The invention also relates to pharmaceutical compositions containing a TNF binding protein and methotrexate useful in such methods.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of TNF-mediateddiseases. More specifically, the present invention relates tocombination therapy for the purpose of preventing or treatingTNF-mediated diseases.

BACKGROUND OF THE INVENTION

[0002] Inflammation is the body's defense reaction to injuries such asthose caused by mechanical damage, infection or antigenic stimulation.An inflammatory reaction may be expressed pathologically wheninflammation is induced by an inappropriate stimulus such as anautoantigen, is expressed in an exaggerated manner or persists wellafter the removal of the injurious agents. Such inflammatory reactionmay include the production of certain cytokines.

[0003] While the etiology of inflammation is poorly understood,considerable information has recently been gained regarding themolecular aspects of inflammation. This research has led toidentification of certain cytokines which are believed to figureprominently in the mediation of inflammation. Cytokines areextracellular proteins that modify the behavior of cells, particularlythose cells that are in the immediate area of cytokine synthesis andrelease. Tumor necrosis factors (TNFs) are a class of cytokines producedby numerous cell types, including monocytes and macrophages.

[0004] At least two TNFs have been previously described, specificallyTNF alpha (TNF-α) and TNF beta (TNF-β or lymphotoxin), and each isactive as a trimeric molecule and is believed to initiate cellularsignaling by crosslinking receptors (Engelmann et al. (1990), J. Biol.Chem., 265:14497-14504).

[0005] Several lines of evidence implicate TNF-α and TNF-β as majorinflammatory cytokines. These known TNFs have important physiologicaleffects on a number of different target cells which are involved ininflammatory responses to a variety of stimuli such as infection andinjury. The proteins cause both fibroblasts and synovial cells tosecrete latent collagenase and prostaglandin E₂ and cause osteocytecells to stimulate bone resorption. These proteins increase the surfaceadhesive properties of endothelial cells for neutrophils. They alsocause endothelial cells to secrete coagulant activity and reduce theirability to lyse clots. In addition they redirect the activity ofadipocytes away from the storage of lipids by inhibiting expression ofthe enzyme lipoprotein lipase. TNFs also cause hepatocytes to synthesizea class of proteins known as “acute phase reactants,” which act on thehypothalamus as pyrogens (Selby et al. (1988), Lancet, 1(8583):483;Starnes, Jr. et al. (1988), J. Clin. Invest., 82:1321; Oliff et al.(1987), Cell, 50:555; and Waage et al. (1987), Lancet, 1(8529) :355).

[0006] A disease or medical condition is considered to be a“TNF-mediated disease” if the spontaneous or experimental disease isassociated with elevated levels of TNF in bodily fluids or in tissuesadjacent to the focus of the disease or indication within the body.TNF-mediated diseases may also be recognized by the following twoconditions: (1) pathological findings associated with a disease can bemimicked experimentally in animals by the administration of TNF and (2)the pathology induced in experimental animal models of the disease canbe inhibited or abolished by treatment with agents which inhibit theaction of TNF. Many TNF-mediated diseases satisfy two of these threeconditions, and others will satisfy all three conditions.

[0007] TNF-mediated diseases such as rheumatoid arthritis and psoriaticarthritis are chronic joint diseases that afflict and disable, tovarying degrees, millions of people worldwide. Rheumatoid arthritis is adisease of articular joints in which the cartilage and bone are slowlyeroded away by a proliferative, invasive connective tissue calledpannus, which is derived from the synovial membrane. The disease mayinvolve peri-articular structures such as bursae, tendon sheaths andtendons as well as extra-articular tissues such as the subcutis,cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles,nervous system (central and peripheral) and eyes (Silberberg (1985),Anderson's Pathology, Kissane (ed.), II:1828).

[0008] It is believed that rheumatoid arthritis results from thepresentation of a relevant antigen to an immunogenetically susceptiblehost. The antigens that could potentially initiate an immune responseresulting in rheumatoid arthritis might be endogenous or exogenous.Possible endogenous antigens include collagen, mucopolysaccharides andrheumatoid factors. Exogenous antigens include mycoplasms, mycobacteria,spirochetes and viruses. By-products of the immune reaction inflame thesynovium (i.e., prostaglandins and oxygen radicals) and triggerdestructive joint changes (i.e., collagenase).

[0009] There is a wide spectrum of disease severity, but many patientsrun a course of intermittent relapses and remissions with an overallpattern of slowly progressive joint destruction and deformity. Theclinical manifestations may include symmetrical polyarthritis ofperipheral joints with pain, tenderness, swelling and loss of functionof affected joints; morning stiffness; and loss of cartilage, erosion ofbone matter and subluxation of joints after persistent inflammation.Extra-articular manifestations include rheumatoid nodules, rheumatoidvasculitis, pleuropulmonary inflammations, scleritis, sicca syndrome,Felty's syndrome (splenomegaly and neutropenia), osteoporosis and weightloss (Katz (1985), Am. J. Med., 79:24 and Krane and Simon (1986),Advances in Rheumatology, Synderman (ed.), 70(2):263-284). The clinicalmanifestations result in a high degree of morbidity resulting indisturbed daily life of the patient.

[0010] Additionally, preclinical results with various predictive animalmodels of rheumatoid arthritis have suggested that inhibition of TNF-αcan have a major impact on disease progression and severity (Dayer etal. (1994), European Cytokine Network, 5(6):563-571 and Feldmann et al.(1995), Annals Of The New York Academy Of Sciences, 66:272-278).Moreover, recent human clinical trials in. rheumatoid arthritis withinhibitors of TNF have shown promising results (Rankin et al. (1995),British Journal Of Rheumatology, 3(4):4334-4342; Elliott et al. (1995),Lancet, 344:1105-1110; Tak et al. (1996), Arthritis and Rheumatism,39:1077-1081; Paleolog et al. (1996), Arthritis and Rheumatism,39:1082-1091 and Moreland et al. (1997), New England Journal ofMedicine, 337:141-147.).

[0011] It is an object of the present invention to provide therapeuticmethods and compositions for the treatment of TNF-mediated diseases.This and other objects of the present invention will become apparentfrom the description hereinafter.

SUMMARY OF THE INVENTION

[0012] The present invention relates to therapies for preventing andtreating TNF-mediated diseases in a patient. The present inventionspecifically relates to combination therapy using a TNF binding proteinfor preventing and treating TNF-mediated diseases, including rheumaticdiseases, and the systemic inflammation and body weight loss associatedtherewith. The type of treatment herein referred to is intended formammals, including humans.

BRIEF DESCRIPTION OF THE FIGURES

[0013] Numerous aspects and advantages of the present invention willbecome apparent upon review of the figures, wherein:

[0014]FIG. 1 depicts a nucleic acid sequence (SEQ ID NO: 1) encodingAsp¹-Thr¹⁶¹, mature recombinant human soluble TNF receptor type I. Alsodepicted is the amino acid sequence (SEQ ID NO: 2) of Asp¹-Thr¹⁶¹. Theamino terminus of the amino acid sequence may be methionylated ornonmethionylated.

[0015]FIG. 2 depicts a nucleic acid sequence (SEQ ID NO: 3) encodingLeu¹-Thr¹⁷⁹, mature recombinant human soluble TNF receptor type II. Alsodepicted is the amino acid sequence (SEQ ID NO: 4) of Leu¹-Thr¹⁷⁹. Theamino terminus of the amino acid sequence may be methionylated ornonmethionylated.

[0016]FIG. 3 depicts the effects of c105 sTNFR-I dumbbell alone,methotrexate alone and the combination of c105 sTNFR-I dumbbell andmethotrexate on joint diameter in the adjuvant arthritic rats in Example1.

[0017]FIG. 4 depicts the effects of c105 sTNFR-I dumbbell alone,methotrexate alone and the combination of c105 sTNFR-I dumbbell andmethotrexate on final paw weights (index of arthritis), splenomegaly(index of systemic inflammation) and body weight change in the adjuvantarthritic rats in Example 1.

[0018]FIG. 5 depicts the final analysis (inhibition at termination) ofthe effects of c105 sTNFR-I dumbbell alone, methotrexate alone and thecombination of c105 sTNFR-I dumbbell and methotrexate on joint diameterin the adjuvant arthritic rats in Example 1.

[0019]FIG. 6 depicts the effects of sTNFR-II/Fc alone, methotrexatealone and the combination of sTNFR-II/Fc with methotrexate on final pawweights (index of arthritis), splenomegaly (index of systemicinflammation) and body weight change in the adjuvant arthritic rats inExample 2.

[0020]FIG. 7 depicts the effects of sTNFR-II/Fc alone, methotrexatealone and the combination of sTNFR-II/Fc with methotrexate on adjuvantarthritic rats in Example 2.

[0021]FIG. 8 depicts the effects of c105 sTNFR-I dumbbell alone, fasfusion protein alone and the combination of c105 sTNFR-I dumbbell andfas fusion protein on LPS/D-Galactosamine lethality in rats in Example3.

DETAILED DESCRIPTION OF THE INVENTION T

[0022] he compositions and methods of the invention includeadministering to a patient afflicted with an inflammatory joint diseasean effective amount of a TNF binding protein in combination with any ofone or more anti-inflammatory drugs or therapies. The preferred patientis human.

[0023] TNF binding proteins are disclosed in the art (EP 308 378, EP 422339, GB 2 218 101, EP 393 438, WO 90/13575, EP 398 327, EP 412 486, WO91/03553, EP 418 014, JP 127,800/1991, EP 433 900, U.S. Pat. No.5,136,021, GB 2 246 569, EP 464 533, WO 92/01002, WO 92/13095, WO92/16221, EP 512 528, EP 526 905, WO 93/07863, EP 568 928, WO 93/21946,WO 93/19777, EP 417 563, WO 95/34326, WO 96/28546, and PCT ApplicationNo. PCT/US97/12244 the disclosures of which are hereby incorporated byreference).

[0024] For example, EP 393 438 and EP 422 339 teach the amino acid andnucleic acid sequences of a soluble TNF receptor type I (also known assTNFR-I or 30 kDa TNF inhibitor) and a soluble TNF receptor type II(also known as sTNFR-II or 40 kDa TNF inhibitor), collectively termed“sTNFRs”, as well as modified forms thereof (e.g., fragments, functionalderivatives and variants). EP 393 438 and EP 422 339 also disclosemethods for isolating the genes responsible for coding the inhibitors,cloning the gene in suitable vectors and cell types, and expressing thegene to produce the inhibitors.

[0025] sTNFR-I and sTNFR-II are members of the nerve growth factor/TNFreceptor superfamily of receptors which includes the nerve growth factorreceptor (NGF), the B cell antigen CD40, 4-1BB, the rat T-cell antigenMRC OX40, the fas antigen, and the CD27 and CD30 antigens (Smith et al.(1990), Science, 248:1019-1023). The most conserved feature amongst thisgroup of cell surface receptors is the cysteine-rich extracellularligand binding domain, which can be divided into four repeating motifsof about forty amino acids and which contains 4-6 cysteine residues atpositions which are well conserved (Smith et al. (1990), supra).

[0026] For purposes of this invention, sTNFRs and modified formsthereof, including polypeptides in which amino acids of sTNFR-I andsTNFR-II have been deleted from (“deletion variants”), inserted into(“addition variants”), or substituted for (“substitution variants”) arecollectively termed “TNFbp(s)”. [Unless otherwise indicated, amino acidnumbering for molecules described herein shall correspond to thatpresented for the mature form of molecule (i.e., minus the signalsequence), as depicted by amino acids Asp¹-Thr¹⁶¹ of SEQ ID NO: 2, withany initial MET in each such sequence being residue number “0”.]

[0027] It will be appreciated by those skilled in the art that manycombinations of deletions, insertions and substitutions (individually orcollectively “variant(s)”) can be made within the amino acid sequencesof the sTNFRs, provided that the resulting molecule is biologicallyactive (e.g., possesses the ability to bind TNF).

[0028] An sTNFR variant(s) may be rapidly screened to assess itsphysical properties. It will be appreciated that such variant(s) willdemonstrate similar TNF inhibiting properties, but not necessarily allof the same properties and not necessarily to the same degree as thecorresponding unmodified sTNFR.

[0029] There are two principal variables in the construction of aminoacid sequence variant(s): the location of the mutation site and thenature of the mutation. In designing variant(s), the location of eachmutation site and the nature of each mutation will depend on thebiochemical characteristic(s) to be modified. Each mutation site can bemodified individually or in series, e.g., by (1) deleting the targetamino acid residue, (2) inserting one or more amino acid residuesadjacent to the located site or (3) substituting first with conservativeamino acid choices and, depending upon the results achieved, then withmore radical selections.

[0030] Amino acid sequence deletions generally range from about 1 to 30amino acid residues, preferably from about 1 to 20 amino acid residues,more preferably from about 1 to 10 amino acid residues and mostpreferably from about 1 to 5 contiguous residues. Amino-terminal,carboxy-terminal and internal intrasequence deletions are contemplated.Deletions within the amino acid sequences of the sTNFRs may be made, forexample, in regions of low homology with the sequences of other membersof the NGF/TNF receptor family. Deletions within the amino acidsequences of the sTNFRs in areas of substantial homology with thesequences of other members of the NGF/TNF receptor family will be morelikely to significantly modify the biological activity. Specifically,the sequence similarity among NGF/TNF receptor family members isparticularly high in the region corresponding to the first two disulfideloops of domain 1, the whole of domain 2, and the first disulfide loopof domain 3 (Banner et al. (1993), Cell, 73:431-445). The number oftotal deletions and/or consecutive deletions preferably will be selectedso as to preserve the tertiary structure in the affected domain, e.g.,cysteine crosslinking.

[0031] EP 393 438 teaches a 40 kDa TNF inhibitor Δ51 and a 40 kDa TNFinhibitor Δ53, which are truncated versions of the full-lengthrecombinant 40 kDa TNF inhibitor protein wherein 51 or 53 amino acidresidues, respectively, at the carboxyl terminus of the mature proteinare removed. Accordingly, a skilled artisan would appreciate that thefourth domain of each of the 30 kDa TNF inhibitor and the 40 kDainhibitor is not necessary for TNF inhibition. In fact various groupshave confirmed this understanding. Domain-deletion derivatives of the 30kDa and 40 kDa TNF inhibitors have been generated, and those derivativeswithout the fourth domain retain full TNF binding activity while thosederivatives without the first, second or third domain, respectively, donot retain TNF binding activity (Corcoran et al. (1994), Eur. J.Biochem., 223:831-840; Chih-Hsueh et al. (1995), The Journal ofBiological Chemistry, 270(6):2874-2878; and Scallon et al. (1995),Cytokine, 7(8) :759-770).

[0032] PCT Application No. PCT/US97/12244 teaches truncated forms ofsTNFR-I and sTNFR-II which do not contain the fourth domain (amino acidresidues Thr¹²⁷-Asn¹⁶¹ of sTNFR-I and amino acid residues Pro¹⁴¹-Thr¹⁷⁹of sTNFR-II); a portion of the third domain (amino acid residuesAsn¹¹¹-Cys¹²⁶ of sTNFR-I and amino acid residues Pro¹²³-Lys¹⁴⁰ ofsTNFR-II); and, optionally, which do not contain a portion of the firstdomain (amino acid residues Asp¹-Cys¹⁹ of sTNFR-I and amino acidresidues Leu¹-Cys³² of sTNFR-II). The truncated sTNFRs of the presentinvention include the proteins represented by the formulaR₁-[Cys¹⁹-Cys¹⁰³]-R₂ and R₄-[Cys³²-Cys¹¹⁵]-R₅. These proteins aretruncated forms of sTNFR-I and sTNFR-II, respectively.

[0033] By “R₁-[Cys¹⁹-Cys¹⁰³]-R₂”0 is meant one or more proteins wherein[Cys¹⁹-Cys¹⁰³] represents residues 19 through 103 of sTNFR-I, the aminoacid residue numbering scheme of which is provided in FIG. 1 tofacilitate the comparison; wherein R₁ represents a methionylated ornonmethionylated amine group of Cys¹⁹ or of amino-terminus amino acidresidue(s) selected from any one of Cys¹⁸ to Asp¹ and wherein R₂represents a carboxy group of Cys¹⁰³ or of carboxy-terminal amino acidresidues selected from any one of Phe¹⁰⁴ to Leu¹¹⁰.

[0034] Exemplary truncated sTNFR-I of the present invention include thefollowing molecules (collectively termed 2.6D sTNFR-I):NH₂-[Asp¹-Cys¹⁰⁵]-COOH (also referred to as sTNFR-I 2.6D/C105);NH₂-[Asp¹-Leu¹⁰⁸]-COOH (also referred to as sTNFR-I 2.6D/C106);NH₂-[Asp¹-Asn¹⁰⁵]-COOH (also referred to as sTNFR-I 2.6D/N105);NH₂-[Tyr⁹-Leu¹⁰⁸]-COOH (also referred to as sTNFR-I 2.3D/d8);NH₂-[Cys¹⁹-Leu¹⁰⁸]-COOH (also referred to as sTNFR-I 2.3D/d18); andNH₂-[Ser¹⁶-Leu¹⁰⁸]-COOH (also referred to as sTNFR-I 2.3D/d15), eithermethionylated or nonmethionylated, and variants and derivatives thereof.

[0035] By “R₃-[Cys³²-Cys¹¹⁵]-R₄” is meant one or more proteins wherein[Cys³²-Cys¹¹⁵] represents residues Cys³² through Cys¹¹⁵ of sTNFR-II, theamino acid residue numbering scheme of which is provided in FIG. 2 tofacilitate the comparison; wherein R₃ represents a methionylated ornonmethionylated amine group of Cys³² or of amino-terminus amino acidresidue(s) selected from any one of Cys³¹ to Leu¹ and wherein R₄represents a carboxy group of Cys¹¹⁵ or of carboxy-terminal amino acidresidue(s) selected from any one of Ala¹¹⁶ to Arg¹²².

[0036] An amino acid sequence addition may include insertions of anamino- and/or carboxyl-terminal fusion ranging in length from oneresidue to one hundred or more residues, as well as internalintrasequence insertions of single or multiple amino acid residues.Internal additions may range generally from about 1 to 20 amino acidresidues, preferably from about 1 to 10 amino acid residues, morepreferably from about 1 to 5 amino acid residues, and most preferablyfrom about 1 to 3 amino acid residues. Additions within the amino acidsequences of the sTNFRs may be made in regions of low homology with thesequences of other members of the NGF/TNF receptor family. Additionswithin the amino acid sequence of the sTNFRs in areas of substantialhomology with the sequences of other members of the NGF/TNF receptorfamily will be more likely to significantly modify the biologicalactivity. Additions preferably include amino acid sequences derived fromthe sequences of the NGF/TNF receptor family members.

[0037] An amino-terminus addition is contemplated to include theaddition of a methionine (for example, as an artifact of the directexpression in bacterial recombinant cell culture). A further example ofan amino-terminal addition includes the fusion of a signal sequence tothe amino-terminus of mature sTNFRs in order to facilitate the secretionof protein from recombinant host cells. Such signal sequences generallywill be obtained from and thus be homologous to the intended host cellspecies. For prokaryotic host cells that do not recognize and processthe native signal sequence of the sTNFRs, the signal sequence may besubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase or heat-stableenterotoxin II leader sequences. For expression in yeast cells thesignal sequence may be selected, for example, from the group of theyeast invertase, alpha factor or acid phosphatase leader sequences. Inmammalian cell expression the native signal sequences (EP 393 438 and EP422 339) are satisfactory, although other mammalian signal sequences maybe suitable, for example sequences derived from other NGF/TNF receptorfamily members.

[0038] An example of an amino- or a carboxy-terminus addition includeschimeric proteins comprising the amino-terminal or carboxy-terminalfusion of a TNFbp(s) with all or part of the constant domain of theheavy or light chain of human immunoglobulin (individually orcollectively, (“sTNFR Fc(s)”). Such chimeric polypeptides are preferredwherein the immunoglobulin portion of each comprises all of the domainsexcept the first domain of the constant region of the heavy chain ofhuman immunoglobulin such as IgG (e.g., IgG1 or IgG3), IgA, IgM or IgE.A skilled artisan will appreciate that any amino acid of theimmunoglobulin portion can be deleted or substituted with one or moreamino acids, or one or more amino acids can be added as long as the TNFbinding protein portion still binds TNF and the immunoglobulin portionshows one or more of its characteristic properties.

[0039] Another group of variant(s) is amino acid substitution variant(s)of the amino acid sequence of sTNFRs. These are variant(s) wherein atleast one amino acid residue in an sTNFR is removed and a differentresidue inserted in its place. Substitution variant(s) include allelicvariant(s) which are characterized by naturally-occurring nucleotidesequence changes in the species population that may or may not result inan amino acid change. One skilled in the art can use any informationknown about the binding or active site of the polypeptide in theselection of possible mutation sites.

[0040] One method for identifying amino acid residues or regions formutagenesis of a protein is called “alanine scanning mutagenesis”, asdescribed by Cunningham and Wells (1989), Science, 244:1081-1085, thedisclosure of which is hereby incorporated by reference. In this method,an amino acid residue or group of target residues is identified (e.g.,charged residues such as Arg, Asp, His, Lys and Glu) and replaced by aneutral or negatively-charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Thosedomains/residues demonstrating functional sensitivity to thesubstitutions are then refined by introducing additional or alternateresidues at the sites of substitution. Thus, the site for introducing anamino acid sequence modification is predetermined. To optimize theperformance of a mutation at a given site, alanine scanning or randommutagenesis may be conducted and the variant(s) may be screened for theoptimal combination of desired activity and degree of activity.

[0041] The sites of greatest interest for substitutional mutagenesisinclude sites in which particular amino acid residues within an sTNFRare substantially different from other species or or other NGF/TNFreceptor family members in terms of side-chain bulk, charge and/orhydrophobicity. Other sites of interest include those in whichparticular residues of an sTNFR are identical among other species orother NGF/TNF receptor family members, as such positions are generallyimportant for the biological activity of a protein.

[0042] Other sites of interest include those in which particularresidues are similar or identical with those of such sTNFR-I-likeproteins and sTNFR-II-like proteins. Accordingly, the followinginformation has been elucidated concerning sTNFR-I (Banner et al.(1993), supra, and Fu et al. (1995), Protein Engineering,8(12):1233-1241). Residues Tyr⁹, Thr³⁹, His⁵⁵ in Domain 1, residuesPhe⁴⁹, Ser⁶³, Asp⁸² in Domain 2 and residues Tyr⁹² and Ser¹⁰⁷ in Domain3 have been identified as being potentially important for thestabilization of the structure of Domains 1, 2 and 3, respectively.Residues Pro¹² and His⁵⁵ have been identified as potentially interactingwith Ser⁸⁶-Tyr⁸⁷ on subunit C of TNFα. Residues Glu⁴⁵-Phe⁴⁹ have beenidentified as being in a loop which potentially interacts with residuesLeu²⁹-Arg³² of TNF-α subunit A. Residues Gly⁴⁸ has been identified aspotentially interacting with Asn¹⁹-Pro²⁰ on subunit A of TNF-α. ResidueHis⁵⁸-Leu⁶⁰ have been identified as being in an extended strandconformation and side chain interactions with residues Arg³¹-Ala³³ onsubunit A of TNFα have been potentially identified with residue His⁵⁸ ofsTNFR-I specifically interacting with residue Arg³¹. ResiduesLys⁶⁴-Arg⁶⁶ have been identified as being in an extended strandconformation and have been identified as having side chain and mainchain interactions with residues Ala¹⁴⁵-Glu¹⁴⁶ and residue Glu⁴⁶ onsubunit A of TNF-α. Residue Met⁶⁹ has been identified as potentiallyinteracting with residue Tyr115 on subunit A of TNF-α. ResiduesHis⁹⁴-Phe¹⁰¹ have been identified as forming a loop which interacts withresidues Thr⁷²-Leu⁷⁵ and Asn¹³⁷ of subunit C of TNF-α, with residueTrp⁹⁶ of sTNFR-I specifically interacting with residues Ser⁷¹-Thr⁷² onsubunit C of TNF-α, Leu¹⁰⁰ of sTNFR-I being in close proximity withresidue Asn¹³⁷ on subunit C of TNF-α and residue Gln¹⁰² of sTNFR-Ispecifically interacting with residue Pro¹¹³ on subunit A of TNF-α.

[0043] In addition to the cysteines forming the 3 pairs of disulfidebonds within each of the four domains of the molecule, there are severalother conserved residues that contribute to the stabilization of thetertiary fold of each domain.

[0044] There are two main classes into which these stabilizing residuesfall. The first type contributes to the shielding of the disulfide bondsulfur atoms from solvent. An example of this residues in domain 3 isTyr⁹². In domain 4 Phe¹³³ helps to shield the Cys¹²⁸-Cys¹³⁹ disulfidebond. All four domains have either a Tyr or Phe at these samestructurally conserved locations. The second class of stabilizingresidues form hydrogen bonds within their respective domains. Withindomain 3 Asn¹²³ and Ser¹⁰⁷ form a hydrogen bond and Ser¹⁰⁷ forms anadditional hydrogen bond with Thr¹²⁴. For domain 4 these residuesinclude Asn¹⁴⁴ and Ser¹⁴¹.

[0045] In addition there are hydrogen bonds between domain 3 and 4 thatare not seen between other domains. These hydrogens bonds are (1) Asn¹⁰⁵main-chain oxygen and Asn¹³⁷ side-chain nitrogen and (2) Ser¹⁰⁷side-chain oxygen and Asn¹³⁷ main-chain nitrogen.

[0046] A skilled artisan will appreciate that initially the sites shouldbe modified by substitution in a relatively conservative manner. Suchconservative substitutions are shown in Table 1 under the heading of“Preferred Substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes (ExemplarySubstitutions) may be introduced and/or other additions/deletions may bemade and the resulting products screened. TABLE 1 Amino AcidSubstitutions Original Preferred Exemplary Residue SubstitutionsSubstitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn(N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) AsnAsn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L) Ile norleucine; Ile;Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; IlePhe (F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S) Thr Thr Thr (T)Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile;Leu; Met; Phe; Ala; norleucine

[0047] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle (1982), J. Mol. Biol.,157:105-131, the disclosure of which is incorporated herein byreference). It is known that certain amino acids may be substituted forother amino acids having a similar hydropathic index or score and stillretain a similar biological activity.

[0048] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity,particularly where the functionally equivalent protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. U.S. Pat. No. 4,554,101, the disclosure of which isincorporated herein by reference, states that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

[0049] U.S. Pat. No. 4,554,101 also teaches the identification andpreparation of epitopes from primary amino acid sequences on the basisof hydrophilicity. Through the methods disclosed in U.S. Pat. No.4,554,101 a skilled artisan would be able to identify epitopes, forexample, within the amino acid sequence of an sTNFR. These regions arealso referred to as “epitopic core regions”. Numerous scientificpublications have been devoted to the prediction of secondary structure,and to the identification of epitopes, from analyses of amino acidsequences (Chou and Fasman (1974), Biochemistry, 13(2):222-245; Chou andFasman (1974), Biochemistry, 13(2):211-222; Chou and Fasman (1978), Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148; Chou and Fasman (1978),Ann. Rev. Biochem., 47:251-276 and Chou and Fasman (1979), Biophys. J.,26:367-384, the disclosures of which are incorporated herein byreference). Moreover, computer programs are currently available toassist with predicting antigenic portions and epitopic core regions ofproteins. Examples include those programs based upon the Jameson-Wolfanalysis (Jameson and Wolf (1988), Comput. Appl. Biosci., 4(l):181-186and Wolf et al. (1988), Comput. Appl. Biosci., 4(1):187-191, thedisclosures of which are incorporated herein by reference); the programPepPlot® (Brutlag et al. (1990), CABS, 6:237-245 and Weinberger et al.(1985), Science, 228:740-742, the disclosures of which are incorporatedherein by reference); and other programs for protein tertiary structureprediction (Fetrow and Bryant (1993), BIOTECHNOLOGY, 11:479-483, thedisclosure of which is incorporated herein by reference).

[0050] In contrast, substantial modifications in the functional and/orchemical characteristics of the sTNFRs may be accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) therelative charge or hydrophobicity of the protein at the target site or(c) the bulk of the side chain. Naturally-occurring residues are dividedinto groups based on common side chain properties:

[0051] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0052] 2) neutral hydrophilic: Cys, Ser, Thr;

[0053] 3) acidic: Asp, Glu;

[0054] 4) basic: Asn, Gln, His, Lys, Arg;

[0055] 5) aromatic: Trp, Tyr, Phe; and

[0056] 6) residues that influence chain orientation:

[0057] Gly, Pro.

[0058] Non-conservative substitutions may involve the exchange of amember of one of these groups for another. Such substituted residues maybe introduced into regions of the sTNFRs that, for example, arehomologous with other NGF/TNF receptor family members or intonon-homologous regions of the protein.

[0059] A variety of amino acid substitutions or deletions may be made tomodify or add N-linked or O-linked glycosylation sites, resulting in aprotein with altered glycosylation. The sequence may be modified to addglycosylation sites to or to delete N-linked or O-linked glycosylationsites from the sTNFRs. An asparagine-linked glycosylation recognitionsite comprises a tripeptide sequence which is specifically recognized byappropriate cellular glycosylation enzymes. These tripeptide sequencesare either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acidother than Pro. Proven or predicted asparagine residues of 30 kDa TNFinhibitor exist at positions 14, 105 and 111.

[0060] Specific mutations of the sequences of the sTNFRs may involvesubstitution of a non-native amino acid at the amino-terminus,carboxy-terminus or at any site of the protein that is modified by theaddition of an N-linked or O-linked carbohydrate. Such modifications maybe of particular utility in the addition of an amino acid (e.g.,cysteine), which is advantageous for the linking of a water solublepolymer to form a derivative. For example, WO 92/16221 describes thepreparation of sTNFR-I muteins, e.g., wherein an asparagine residue atposition 105 of the native human protein is changed to cysteine (c105sTNFR-I).

[0061] In a specific embodiment, a variant polypeptide will preferablybe substantially homologous to the amino acid of the sTNFR from which itis derived. The term “substantially homologous” as used herein means adegree of homology that is in excess of 80%, preferably in excess of90%, more preferably in excess of 95% or most preferably even 99%. Thepercentage of homology as described herein is calculated as thepercentage of amino acid residues found in the smaller of the twosequences which align with identical amino acid residues in the sequencebeing compared when four gaps in a length of 100 amino acids may beintroduced to assist in that alignment, as set forth by Dayhoff (1972),Atlas of Protein Sequence and Structure, 5:124, National BiochemicalResearch Foundation, Washington, D.C., the disclosure of which is herebyincorporated by reference. Also included within the term “substantiallyhomologous” are variant(s) of sTNFRs which may be isolated by virtue ofcross-reactivity with antibodies to the amino acid sequences of SEQ IDNO: 2 and SEQ ID NO: 4 or whose genes may be isolated throughhybridization with the DNA of SEQ ID NO: 1 and SEQ ID NO: 3 or withsegments thereof.

[0062] Polypeptide Derivatives

[0063] Chemically-modified derivatives of the TNFbp(s) in which theprotein is linked to a polymer in order to modify properties of theprotein (referred herein as “derivatives”) are included within the scopeof the present invention. Such derivatives may be prepared by oneskilled in the art given the disclosures herein. Conjugates may beprepared using glycosylated, non-glycosylated or de-glycosylatedTNFbp(s) and suitable chemical moieties. Typically non-glycosylatedproteins and water soluble polymers will be used.

[0064] Water soluble polymers are desirable because the protein to whicheach is attached will not precipitate in an aqueous environment, such asa physiological environment. Preferably, the polymer will bepharmaceutically acceptable for the preparation of a therapeutic productor composition. One skilled in the art will be able to select thedesired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically and, if so, thetherapeutic profile of the protein (e.g., duration of sustained release;resistance to proteolysis; effects, if any, on dosage; biologicalactivity; ease of handling; degree or lack of antigenicity and otherknown effects of a water soluble polymer on a therapeutic proteins).

[0065] Suitable, clinically acceptable, water soluble polymers include,but are not limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers (PPG)and other polyalkylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll or dextranand mixtures thereof. As used herein, polyethylene glycol is meant toencompass any of the forms that have been used to derivatize otherproteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water.

[0066] The water soluble polymers each may be of any molecular weightand may be branched or unbranched. Generally, the higher the molecularweight or the more branches, the higher the polymer:protein ratio. Thewater soluble polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each water soluble polymer preferably is betweenabout 5 kDa and about 40 kDa, more preferably between about 10 kDa andabout 35 kDa and most preferably between about 15 kDa and about 30 kDa.—

[0067] There are a number of attachment methods available to thoseskilled in the art, including acylation reactions or alkylationreactions (preferably to generate an amino-terminal chemically modifiedprotein) with a reactive water soluble molecule. See, for example, EP 0401 384; Malik et al. (1992), Exp. Hematol., 20:1028-1035; Francis(1992), Focus on Growth Factors, 3(2):4-10, published by Mediscript,Mountain Court, Friern Barnet Lane, London N20 OLD, UK; EP 0 154 316; EP0 401 384; WO 92/16221; WO 95/34326; WO 95/13312; WO 96/11953; WO96/19459 and WO 96/19459 and the other publications cited herein thatrelate to pegylation, the disclosures of which are hereby incorporatedby reference.

[0068] A specific embodiment of the present invention is an unbranchedmonomethoxy-polyethylene glycol aldehyde molecule having an averagemolecular weight of either about 20 kDa or about 33 kDa (e.g., between30 kDa and 35 kDa), or a tertiary-butyl polyethylene glycol aldehydehaving an average molecular weight of about 33 kDa (e.g., between 30 kDaand 35 kDa) conjugated via reductive alkylation to the TNFbp(s).

[0069] The pegylation also may be specifically carried out using watersoluble polymers having at least one reactive hydroxy group (e.g.polyethylene glycol). The water soluble polymer can be reacted with anactivating group, thereby forming an “activated linker” useful inmodifying various proteins. The activated linkers can be monofunctional,bifunctional, or multifunctional.

[0070] Activating groups which can be used to link the water solublepolymer to two or more proteins include the following: sulfone,maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxiraneand 5-pyridyl. Useful reagents having a reactive sulfone group that canbe used in the methods include, without limitation, chlorosulfone,vinylsulfone and divinylsulfone. These PEG derivatives are stableagainst hydrolysis for extended-periods in aqueous environments at pHsof about 11 or less, and can form linkages with molecules to formconjugates which are also hydrolytically stable. Two particularly usefulhomobifunctional derivatives are PEG-bis-chlorosulfone andPEG-bis-vinylsulfone (WO 95/13312).

[0071] WO 97/04003, the disclosure of which is hereby incorporated byreference, teaches methods of making sulfone-activated linkers byobtaining a compound having a reactive hydroxyl group and converting thehydroxyl group to a reactive Michael acceptor to form an activatedlinker, with the use of tetrahydrofuran (THF) as the solvent for theconversion. The application also teaches a process for purifying theactivated linkers which utilizes hydrophobic interaction chromatographyto separate the linkers based on size and end-group functionality.

[0072] Polyvalent Forms

[0073] Polyvalent forms, i.e., molecules comprising more than one activemoiety, may be constructed. In one embodiment, the molecule may possessmultiple tumor necrosis factor binding sites for the TNF ligand.Additionally, the molecule may possess at least one tumor necrosisfactor binding site and, depending upon the desired characteristic ofpolyvalent form, at least one site of another molecule (e.g., aTNFbp(s), and an interleukin-1 receptor antagonist (“IL-1ra”) asdescribed below).

[0074] In one embodiment, the polyvalent form may be constructed, forexample, by chemically coupling at least one TNFbp(s) and another moietywith any clinically accepted linker (e.g., a water-soluble polymer). Inprinciple the linker must not impart new immunogenecity nor, by virtueof the new amino acid residues, alter the hydrophobicity and chargebalance of the structure which affects its biodistribution andclearance.

[0075] The water soluble polymers can be, based on the monomers listedherein, homopolymers, random or block copolymers, terpolymers straightchain or branched, substituted or unsubstituted. The polymer can be ofany length or molecular weight, but these characteristics can affect thebiological properties. Polymer average molecular weights particularlyuseful for decreasing clearance rates in pharmaceutical applications arein the range of 2,000 to 35,000 daltons. In addition, the length of thepolymer can be varied to optimize or confer the desired biologicalactivity.

[0076] The active moieties may be linked using conventional couplingtechniques (see WO 92/16221, WO 95/13312 and WO 95/34326, thedisclosures of which are hereby incorporated by reference). For example,WO 92/16221 and WO 95/34326 describe the preparation of variousdimerized sTNFR-I molecules, e.g., dimerized c105 sTNFR-I.

[0077] Alternatively, a bivalent molecule may consist of two tandemrepeats of sTNFRs separated by a polypeptide linker region. The designof the polypeptide linkers is similar in design to the insertion ofshort loop sequences between domains in the de novo design of proteins(Mutter (1988), TIBS, 13:260-265 and Regan and DeGrado (1988), Science,241:976-978, the disclosures of which are hereby incorporated byreference). Several different linker constructs have been assembled andshown to be useful for forming single chain antibodies; the mostfunctional linkers vary in size from 12 to 25 amino acids (amino acidshaving unreactive side groups, e.g., alanine, serine and glycine) whichtogether constitute a hydrophilic sequence, have a few oppositelycharged residues to enhance solubility and are flexible (Whitlow andFilpula (1991), Methods: A Companion to Methods in Enzymology, 2:97-105;and Brigido et al. (1993), J. Immunol., 150:469-479, the disclosures ofwhich are hereby incorporated by reference). It has been shown that alinker suitable for single chain antibodies is effective to produce adimeric form of the human sTNFR-II (Neve et al. (1996), Cytokine,8(5):365-370, the disclosure of which is hereby incorporated byreference).

[0078] Additionally, a TNFbp(s) may be chemically coupled to biotin, andthe resulting conjugate may then be allowed to bind to avidin, resultingin tetravalent avidin/biotin/TNFbp(s) molecules. A TNFbp(s) may also becovalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) andthe resulting conjugates precipitated with anti-DNP or anti-TNP-IgM toform decameric conjugates.

[0079] In yet another embodiment, recombinant fusion proteins may alsobe produced wherein each recombinant chimeric molecule has a TNFbp(s)sequence amino-terminally or carboxy-terminally fused to all or part ofthe constant domains, but at least one constant domain, of the heavy orlight chain of human immunoglobulin. For example, a chimericTNFbp(s)/IgG1 (or IgG1/TNFbp(s)) fusion protein may be produced from alight chain-containing chimeric gene: a TNFbp(s)/human kappa light chainchimera (TNFbp(s)/Ck) or a human kappa light chain/TNFbp(s) chimera(Ck/TNFbp(s)); or a heavy chain-containing chimeric gene: aTNFbp(s)/human gamma-1 heavy chain chimera (TNFbp(s)/Cg-1) or a humangamma-1 heavy chain/TNFbp(s) chimera (Cg-1/TNFbp(s)). Followingtranscription and translation of a heavy-chain chimeric gene, or of alight chain-containing gene and a heavy-chain chimeric gene, the geneproducts may be assembled into a single chimeric molecule having aTNFbp(s) displayed bivalently. Additional details relating to theconstruction of such chimeric molecules are disclosed in U.S. Pat. No.5,116,964, WO 89/09622, WO 91/16437 and EP 315062, the disclosures ofwhich are hereby incorporated by reference.

[0080] In yet a further embodiment, recombinant fusion proteins may alsobe produced wherein each recombinant chimeric molecule has at least oneTNFbp(s), as described herein, and at least a portion of the region186-401 of osteoprotogerin, as described in European Patent ApplicationNo. 96309363.8, the disclosures of which are hereby incorporated byreference. Either the TNFbp(s) or the portion of osteoprotogerin may beat the amino-terminus or the carboxy-terminus of the chimeric molecule.

[0081] Synthesis of TNFbp(s)

[0082] The production of TNFbp(s) is described in further detail below.Such proteins may be prepared, for example, by recombinant techniques orby in vitro chemical synthesis.

[0083] Polynucleotides

[0084] Based upon the present description and using the universal codontable, one of ordinary skill in the art can readily determine all of thenucleic acid sequences which encode the amino acid sequence of theTNFbp(s).

[0085] Recombinant expression techniques conducted in accordance withthe descriptions set forth below may be followed to produce each suchpolynucleotide and to express the encoded proteins. For example, byinserting a nucleic acid sequence which encodes a TNFbp(s) into anappropriate vector, one skilled in the art can readily produce largequantities of the desired nucleotide sequence. The sequences can then beused to generate detection probes or amplification primers.Alternatively, a polynucleotide encoding a TNFbp(s) can be inserted intoan expression vector. By introducing the expression vector into anappropriate host, the desired protein may be produced in large amounts.

[0086] As further described herein, there are numerous host/vectorsystems available for the propagation of desired nucleic acid sequencesand/or the production of the desired proteins. These include, but arenot limited to, plasmid, viral and insertional vectors, and prokaryoticand eukaryotic hosts. One skilled in the art can adapt a host/vectorsystem which is capable of propagating or expressing heterologous DNA toproduce or express the sequences of the present invention.

[0087] Furthermore, it will be appreciated by those skilled in the artthat, in view of the present disclosure, the nucleic acid sequenceswithin the scope of the present invention include the nucleic acids ofFIGS. 1 and 3, as well as degenerate nucleic acid sequences thereof,nucleic acid sequences which encode variant(s) of the sTNFRs, and thosenucleic acid sequences which hybridize to complements of the nucleicacids of FIGS. 1 and 3 under hybridization conditions, or equivalentconditions thereto, disclosed in the cDNA library screening sectionbelow.

[0088] Also provided by the present invention are recombinant DNAconstructs involving vector DNA together with the DNA sequences encodingthe desired proteins. In each such DNA construct, the nucleic acidsequence encoding a desired protein (with or without signal peptides) isin operative association with a suitable expression control orregulatory sequence capable of directing the replication and/orexpression of the desired protein in a selected host.

[0089] Recombinant Expression

[0090] Preparation of Polynucleotides

[0091] A nucleic acid sequence encoding a TNFbp(s) can readily beobtained in a variety of ways including, without limitation, chemicalsynthesis, cDNA or genomic library screening, expression libraryscreening and/or PCR amplification of cDNA. These methods and otherswhich are useful for isolating such nucleic acid sequences are set forthin Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; by Ausubel etal. (1994), Current Protocols in Molecular Biology, Current ProtocolsPress; and by Berger and Kimmel (1987), Methods in Enzymology: Guide toMolecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego,Calif., the disclosures of which are hereby incorporated by reference.

[0092] Chemical synthesis of a nucleic acid sequence which encodes adesired protein can be accomplished using methods well known in the art,such as those set forth by Engels et al. (1989), Angew. Chem. Intl. Ed.,28:716-734 and Wells et al. (1985), Gene, 34:315, the disclosures ofwhich is hereby incorporated by reference. These methods include, interalia, the phosphotriester, phosphoramidite and H-phosphonate methods ofnucleic acid sequence synthesis. Large nucleic acid sequences, forexample those larger than about 100 nucleotides in length, can besynthesized as several fragments. The fragments can then be ligatedtogether to form a suitable nucleic acid sequence. A preferred method ispolymer-supported synthesis using standard phosphoramidite chemistry.

[0093] Alternatively, a suitable nucleic acid sequence may be obtainedby screening an appropriate cDNA library (i.e., a library prepared fromone or more tissue sources believed to express the protein) or a genomiclibrary (a library prepared from total genomic DNA). The source of thecDNA library is typically a tissue or cell source from any species thatis believed to express a desired protein in reasonable quantities. Thesource of the genomic library may be any tissue or tissues from anymammalian or other species believed to harbor a gene encoding a desiredprotein.

[0094] Each hybridization medium can be screened for the presence of aDNA encoding a desired protein using one or more nucleic acid probes(oligonucleotides, cDNA or genomic DNA fragments that possess anacceptable level of homology to the cDNA or gene to be cloned) that willhybridize selectively with cDNA(s) or gene(s) present in the library.The probes typically used for such screening encode a small region ofDNA sequence from the same or a similar species as the species fromwhich the library is prepared. Alternatively, the probes may bedegenerate, as discussed herein.

[0095] Hybridization is typically accomplished by annealing theoligonucleotide probe or cDNA to the clones under conditions ofstringency that prevent non-specific binding but permit binding of thoseclones that have a significant level of homology with the probe orprimer. Typical hybridization and washing stringency conditions dependin part on the size (i.e., number of nucleotides in length) of the cDNAor oligonucleotide probe and whether the probe is degenerate. Theprobability of identifying a clone is also considered in designing thehybridization medium (e.g., whether a cDNA or genomic library is beingscreened).

[0096] Where a DNA fragment (such as cDNA) is used as a probe, typicalhybridization conditions include those as set forth in Ausubel et al.(1994), supra. After hybridization, the hybridization medium is washedat a suitable stringency, depending on several factors such as probesize, expected homology of probe to clone, the hybridization mediumbeing screened, the number of clones being screened and the like.

[0097] Exemplary stringent hybridization conditions are hybridization in6×SSC at 62-67° C., followed by washing in 0.1×SSC at 62-67° C. forapproximately one hour. Alternatively, exemplary stringent hybridizationconditions are hybridization at 45-55% formamide, 6×SSC at 40-45° C.,followed by washing in 0.1×SSC at 62-67° C. for approximately one hour.Also included are DNA sequences which hybridize to the nucleic acidsequences set forth in FIGS. 1 and 3 under relaxed hybridizationconditions and which encode a TNFbp(s). Examples of such relaxedstringency hybridization conditions are 6×SSC at 45-55° C. orhybridization with 30-40% formamide at 40-45° C., followed by washing in1-2×SSC at 55° C. for approximately 30 minutes. See Maniatis et al.(1982), Molecular Cloning (A Laboratory Manual), Cold Spring HarborLaboratory, pages 387 to 389, the disclosure of which is herebyincorporated by reference.

[0098] There are also exemplary protocols for stringent washingconditions where oligonucleotide probes are used to screen hybridizationmedia. For example, a first protocol uses 6×SSC with 0.05 percent sodiumpyrophosphate at a temperature of between about 35° C. and 63° C.,depending on the length of the probe. For example, 14 base probes arewashed at 35-40° C., 17 base probes at 45-50° C., 20 base probes at52-57° C., and 23 base probes at 57-63° C. The temperature can beincreased 2-3° C. where the background non-specific binding appearshigh. A second protocol uses tetramethylammonium chloride (TMAC) forwashing. One such stringent washing solution is 3 M TMAC, 50 mMTris-HCl, pH 8.0 and 0.2% SDS.

[0099] Another method for obtaining a suitable nucleic acid sequenceencoding a TNFbp(s) is the polymerase chain reaction (PCR). In thismethod, cDNA is prepared from poly(A)+RNA or total RNA using the enzymereverse transcriptase. Two primers, typically complementary to twoseparate regions of cDNA (oligonucleotides) encoding the desiredprotein, are then added to the cDNA along with a polymerase such as Taqpolymerase and the polymerase amplifies the cDNA region between the twoprimers.

[0100] The oligonucleotide sequences selected as probes or primersshould be of adequate length and sufficiently unambiguous so as tominimize the amount of non-specific binding that may occur duringscreening or PCR amplification. The actual sequence of the probes orprimers is usually based on conserved or highly homologous sequences orregions. Optionally, the probes or primers can be fully or partiallydegenerate, i.e., can contain a mixture of probes/primers, all encodingthe same amino acid sequence but using different codons to do so. Analternative to preparing degenerate probes is to place an inosine insome or all of those codon positions that vary by species. Theoligonucleotide probes or primers may be prepared by chemical synthesismethods for DNA, as described herein.

[0101] Vectors

[0102] DNA encoding the desired proteins may be inserted into vectorsfor further cloning (amplification of the DNA) or for expression.Suitable vectors are commercially available or may be specificallyconstructed. The selection or construction of an appropriate vector willdepend on (1) whether it is to be used for DNA amplification or for DNAexpression, (2) the size of the DNA to be inserted into the vector and(3) the intended host cell to be transformed with the vector.

[0103] The vectors each typically involve a nucleic acid sequence whichencodes a desired protein operatively linked to one or more of thefollowing expression control or regulatory sequences capable ofdirecting, controlling or otherwise effecting the expression of adesired protein by a selected host cell. Each vector contains variouscomponents, depending on its function (amplification of DNA orexpression of DNA) and its compatibility with the intended host cell.The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more selection or marker genes, a promoter, an enhancer element, atranscription termination sequence and the like. These components may beobtained from natural sources or be synthesized by known procedures.

[0104] Examples of suitable prokaryotic cloning vectors includebacteriophages such as lambda derivatives, or plasmids from E. coli(e.g. pBR322, col E1, pUC, the F-factor and Bluescript® plasmidderivatives (Stratagene, La Jolla, Calif.)). Other appropriateexpression vectors, of which numerous types are known in the art for thehost cells described below, can also be used for this purpose.

[0105] Signal Sequence

[0106] The nucleic acid encoding a signal sequence may be inserted 5′ ofthe sequence encoding a desired protein, e.g, it may be a component of avector or it may be a part of a nucleic acid encoding a desired protein.The nucleic acids encoding the native signal sequences of the sTNFRs areknown (EP 393 438, EP 422 339 and WO 96/28546, the disclosures of whichare hereby incorporated by reference).

[0107] Origin of Replication

[0108] Expression and cloning vectors each generally include a nucleicacid sequence that enables the vector to replicate in one or moreselected host cells. In a cloning vector, this sequence is typically onethat enables the vector to replicate independently of the hostchromosomal DNA and includes an origin of replication or autonomouslyreplicating sequence. Such sequences are well known. The origin ofreplication from the plasmid pBR322 is suitable for most Gram-negativebacteria, and various origins (e.g., Simian Virus 40 (SV40), polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication is not needed for mammalianexpression vectors (for example, the SV40 origin is often used onlybecause it contains the early promoter).

[0109] Selection Gene

[0110] The expression and cloning vectors each typically contain aselection gene. This gene encodes a “marker” protein necessary for thesurvival or growth of the transformed host cells when grown in aselective culture media. Host cells that are not transformed with thevector will not contain the selection gene and, therefore, will notsurvive in the culture media. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate or tetracycline; (b) complementauxotrophic deficiencies; or (c) supply critical nutrients not availablefrom the culture media.

[0111] Other selection genes may be used to amplify the genes to beexpressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Thecell transformants are placed under selection pressure which only thetransformants are uniquely adapted to survive by virtue of the markerbeing present in the vector. Selection pressure is imposed by culturingthe transformed cells under conditions in which the concentration ofselection agent in the media is successively changed, thereby leading toamplification of both the selection gene and the DNA that encodes thedesired protein. As a result, increased quantities of the desiredprotein are synthesized from the amplified DNA.

[0112] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedia that contains methotrexate, a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is used is the Chinese hamsterovary cell line deficient in DHFR activity (Urlaub and Chasin (1980),Proc. Natl. Acad. Sci., USA, 77(7):4216-4220, the disclosure of which ishereby incorporated by reference). The transformed cells are thenexposed to increased levels of methotrexate. This leads to the synthesisof multiple copies of the DHFR gene and, concomitantly, multiple copiesof other DNA present in the expression vector, such as the DNA encodinga desired protein.

[0113] Promoter

[0114] Expression and cloning vectors each will typically contain apromoter that is recognized by the host organism and is operably linkedto a nucleic acid sequence encoding the desired protein. A promoter isan untranslated sequence located upstream (5′) to the start codon of astructural gene (generally within about 100 to 1000 bp) that controlsthe transcription and translation of a particular nucleic acid sequence.A promoter may be conventionally grouped into one of two classes,inducible promoters and constitutive promoters. An inducible promoterinitiates increased levels of transcription from DNA under its controlin response to some change in culture conditions, such as the presenceor absence of a nutrient or a change in temperature. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A promoter may be operably linked to DNA encoding a desiredprotein by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence. The nativepromoter sequences of sTNFRs may be used to direct amplification and/orexpression of the DNA encoding a desired protein. A heterologouspromoter is preferred, however, if it permits greater transcription andhigher yields of the expressed protein as compared to the nativepromoter and if it is compatible with the host cell system that has beenselected for use. For example, any one of the native promoter sequencesof other NGF/TNF receptor family members may be used to directamplification and/or expression of the DNA encoding a desired protein.

[0115] Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase; atryptophan (trp) promoter system; a bacterial luminescence (luxR) genesystem and hybrid promoters such as the tac promoter. Other knownbacterial promoters are also suitable. Their nucleotide sequences havebeen published, thereby enabling one skilled in the art to ligate eachselected sequence to the desired DNA sequence using linkers or adaptorsas needed to supply any required restriction sites.

[0116] Suitable promoter sequences for use with yeast hosts are alsowell known in the art. Suitable promoters for use with mammalian hostcells are well known and include those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and, most preferably,SV40. Other suitable mammalian promoters include heterologous mammalianpromoters, e.g., heat-shock promoters and the actin promoter.

[0117] Enhancer Element

[0118] The expression and cloning vectors each will typically contain anenhancer sequence to increase the transcription by higher eukaryotes ofa DNA sequence encoding a desired protein. Enhancers are cis-actingelements of DNA, usually from about 10-300 bp in length, that act on thepromoter to increase its transcription. Enhancers are relativelyorientation and position independent. They have been found 5′ and 3′ tothe transcription unit. Yeast enhancers are advantageously used withyeast promoters. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Additionally, viral enhancers such as the SV40 enhancer, thecytomegalovirus early promoter enhancer, the polyoma enhancer andadenovirus enhancers are exemplary enhancing elements for the activationof eukaryotic promoters. While an enhancer may be spliced into a vectorat a position 5′ or 3′ to a DNA encoding a desired protein, it istypically located at a site 5′ from the promoter.

[0119] Transcription Termination

[0120] Expression vectors used in eukaryotic host cells each willtypically contain a sequence necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and occasionally 3′ untranslated regions ofeukaryotic DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA encoding a desired protein.

[0121] Vector Construction

[0122] The construction of a suitable vector containing one or more ofthe herein-listed components (together with the desired coding sequence)may be accomplished by standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored and religated in the desiredorder to generate the required vector. To confirm that the correctsequence has been constructed, the ligation mixture may be used totransform E. coli, and successful transformants may be selected by knowntechniques as described herein. Quantities of the vector from thetransformants are then prepared, analyzed by restriction endonucleasedigestion and/or sequenced to confirm the presence of the desiredconstruct.

[0123] A vector that provides for the transient expression of DNAencoding a desired protein in mammalian cells may also be used. Ingeneral, transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of the desired protein encoded by the expressionvector. Each transient expression system, comprising a suitableexpression vector and a host cell, allows for the convenient positiveidentification of proteins encoded by cloned DNAs, as well as for therapid screening of such proteins for desired biological or physiologicalproperties.

[0124] Host Cells

[0125] Any of a variety of recombinant host cells, each of whichcontains a nucleic acid sequence for use in expressing a desiredprotein, is also provided by the present invention. Exemplaryprokaryotic and eukaryotic host cells include bacterial, mammalian,fungal, insect, yeast or plant cells.

[0126] Prokaryotic host cells include, but are not limited to,eubacteria such as Gram-negative or Gram-positive organisms (e.g., E.coli (HB101, DH5a, DH10, and MC1061); Bacilli spp. such as B. subtilis;Pseudomonas spp. such as P. aeruginosa; Streptomyces spp.; Salmonellaspp. such as S. typhimurium; or Serratia spp. such as S. marcescans. Ina specific embodiment, a desired protein may be expressed in E. coli.

[0127] In addition to prokaryotic host cells, TNFbp(s) may be expressedin glycosylated form by any one of a number of suitable host cellsderived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture might be used, whether such cultureinvolves vertebrate or invertebrate cells, including plant and insectcells. Eukaryotic microbes such as filamentous fungi or yeast may besuitable hosts for the expression of a desired protein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms, but a number of other genera,species and strains are well known and commonly available.

[0128] Vertebrate cells may be used, as the propagation of vertebratecells in culture (tissue culture) is a well-known procedure. Examples ofuseful mammalian host cell lines include, but are not limited to, monkeykidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line(293 cells or 293 cells subcloned for growth in suspension culture),baby hamster kidney cells and Chinese hamster ovary cells. Othersuitable mammalian cell lines include, but are not limited to, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, andBHK or HaK hamster cell lines. In a specific embodiment, a desiredprotein may be expressed in COS cells or in baculovirus cells.

[0129] A host cell may be transfected and preferably transformed with adesired nucleic acid under appropriate conditions permitting expressionof the nucleic acid. The selection of suitable host cells and methodsfor transformation, culture, amplification, screening and productproduction and purification are well known in the art (Gething andSambrook (1981), Nature, 293:620-625 or, alternatively, Kaufman et al.(1985), Mol. Cell. Biol., 5(7):1750-1759, or U.S. Pat. No. 4,419,446,the disclosures of which are hereby incorporated by reference). Forexample, for mammalian cells without cell walls, the calcium phosphateprecipitation method may be used. Electroporation, micro-injection andother known techniques may also be used.

[0130] It is also possible that a desired protein may be produced byhomologous recombination or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding a desired protein. Homologous recombination is a techniqueoriginally developed for targeting genes to induce or correct mutationsin transcriptionally-active genes (Kucherlapati (1989), Prog. in Nucl.Acid Res. and Mol. Biol., 36:301, the disclosure of which is herebyincorporated by reference). The basic technique was developed as amethod for introducing specific mutations into specific regions of themammalian genome (Thomas et al. (1986), Cell, 44:419-428; Thomas andCapecchi (1987), Cell, 51:503-512 and Doetschman et al. (1988), Proc.Natl. Acad. Sci., 85:8583-8587, the disclosures of which are herebyincorporated by reference) or to correct specific mutations withindefective genes (Doetschman et al. (1987), Nature, 330:576-578, thedisclosure of which is hereby incorporated by reference). Exemplarytechniques are described in U.S. Pat. No. 5,272,071; WO 92/01069; WO93/03183; WO 94/12650 and WO 94/31560, the disclosures of which arehereby incorporated by reference.

[0131] Culturing the Host Cells

[0132] The method for culturing each of the one or more recombinant hostcells for production will vary depending upon many factors andconsiderations; the optimum production procedure for a given situationwill be apparent to those skilled in the art through minimalexperimentation. Such recombinant host cells are cultured in a suitablemedia and the expressed protein is then optionally recovered, isolatedand purified from the culture media (or from the cell, if expressedintracellularly) by appropriate means known to those skilled in the art.

[0133] Specifically, each of the recombinant cells used to produce adesired protein may be cultured in a culture media suitable for inducingpromoters, selecting suitable recombinant host cells or amplifying thegene encoding the desired protein. The culture media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin or epidermal growth factor), salts (such as sodium chloride,calcium, magnesium and phosphate), buffers (such as HEPES), nucleosides(such as adenosine and thymidine), antibiotics (such as gentamicin),trace elements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or another energysource. Other supplements may also be included, at appropriateconcentrations, as will be appreciated by those skilled in the art.Suitable culture conditions, such as temperature, pH and the like, arealso well known to those skilled in the art for use with the selectedhost cells.

[0134] The resulting expression product may then be purified to nearhomogeneity by using procedures known in the art. Exemplary purificationtechniques are taught in EP 393 438 and EP 422 339, the disclosures ofwhich are hereby incorporated by reference.

[0135] Pharmaceutical Compositions

[0136] The present invention encompasses pharmaceutical preparationseach containing therapeutically- or prophylactically-effective amountsof a TNFbp(s) or a chemically-modified derivative thereof (collectively,“TNFbp product(s)”) in admixture with a vehicle. The vehicle preferablyincludes one or more pharmaceutically and physiologically acceptableformulation materials in admixture with the TNFbp product(s).

[0137] The primary solvent in a vehicle may be either aqueous ornon-aqueous in nature. In addition, the vehicle may containpharmaceutically acceptable excipients for modifying or maintaining thepH preferably between 5-6.5, and more preferably between 5.5-6.0 (e.g.,buffers such as citrates or phosphates, and amino acids such glycine);viscosity; clarity; color; sterility; stability (e.g., sucrose andsorbitol); odor; rate of dissolution (e.g., solubilizers or solubilizingagents such as alcohols, polyethylene glycols and sodium chloride); rateof release; as well as bulking agents for lyophilized formulation (e.g.,mannitol and glycine); surfactants (e.g., polysorbate 20, polysorbate80, triton and pluronics); antioxidants (e.g., sodium sulfite and sodiumhydrogen-sulfite); preservatives (e.g., benzoic acid and salicylicacid); flavoring and diluting agents; emulsifying agents; suspendingagents; solvents; fillers; delivery vehicles and other pharmaceuticaladjuvants and/or excipients. Other effective administration forms suchas parenteral slow-release formulations, inhalant mists, orally-activeformulations, or suppositories are also envisioned. The composition mayalso involve particulate preparations of polymeric compounds such asbulk erosion polymers (e.g., poly(lactic-co-glycolic acid) (PLGA)copolymers, PLGA polymer blends, block copolymers of PEG, and lactic andglycolic acid, poly(cyanoacrylates)); surface erosion polymers (e.g.,poly(anhydrides) and poly(ortho esters)); hydrogel esters (e.g.,pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acidderivatives, alginate, collagen, gelatin, albumin, and starches anddextrans) and composition systems thereof; or preparations of liposomesor microspheres. Such compositions may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent proteins and derivatives. The optimal pharmaceutical formulationfor a desired protein will be determined by one skilled in the artdepending upon the route of administration and desired dosage. Exemplarypharmaceutical compositions are disclosed in Remington's PharmaceuticalSciences, 18th Ed. (1990), Mack Publishing Co., Easton, Pa. 18042, pages1435-1712; Gombotz and Pettit (1995), Bioconjugate Chem., 6:332-351;Leone-Bay, et al. (1995), Journal of Medicinal Chemistry, 38:4263-4269;Haas, et al. (1995), Clinical Immunology and Immunopathology, 76(l):93;WO 94/06457; WO 94/21275; FR 2706772 and WO 94/21235, the disclosures ofwhich are incorporated herein by reference.

[0138] Specific sustained release compositions are available from thefollowing suppliers: Depotech (Depofoam™, a multivesicular liposome) andAlkermes (ProLease™, a PLGA microsphere). Exemplary forms of hyaluronanare disclosed in Peyron and Balazs (1974), Path. Biol., 22(8):731-736;Isdale et al. (1991), J. Drug Dev., 4(2):93-99; Larsen et al. (1993),Journal of Biomedical Materials Research, 27:1129-1134; Namiki, et al.(1982), International Journal of Clinical Pharmacology, Therapy andToxicology, 20(11):501-507; Meyer et al. (1995), Journal of ControlledRelease, 35:67-72; Kikuchi et al. (1996), Osteoarthritis and Cartilage,4:99-110; Sakakibara et al. (1994), Clinical Orthopaedics and RelatedResearch, 299:282-292; Meyers and Brandt (1995), 22(9):1732-1739;Laurent et al. (1995), Acta Orthop Scand, 66(266):116-120; Cascone etal. (1995), Biomaterials, 16(7):569-574; Yerashalmi et al. (1994),Archives of Biochemistry and Biophysics, 313(2):267-273; Bernatchez etal. (1993), Journal of Biomedical Materials Research, 27(5):677-681; Tanet al. (1990), Australian Journal of Biotechnology, 4(1):38-43; Gombotzand Pettit (1995), Bioconjugate Chem., 6:332-351; U.S. Pat. Nos.4,582,865, 4,605,691, 4,636,524, 4,713,448, 4,716,154, 4,716,224,4,772,419, 4,851,521, 4,957,774, 4,863,907, 5,128,326, 5,202,431,5,336,767, 5,356,883; European Patent Application Nos. 0 507 604 A2 and0 718 312 A2; and WO 96/05845, the disclosures of which are herebyincorporated by reference. Specific hyaluronan compositions areavailable from the following suppliers: BioMatrix, Inc. Ridgefield, N.J.(Synvisc™, a 90:10 mixture of a hylan fluid and hylan gel); FidiaS.p.A., Abano Terme, Italy (Hyalgan™, the sodium salt of a roostercomb-derived hyaluronic acid (˜500,000 to ˜700,000 MW)); KakenPharmaceutical Co., Ltd., Tokyo, Japan (Artz™, a 1% solution of arooster-comb derived hyaluronic acid, ˜700,000 MW); Pharmacia AB,Stockholm, Sweden (Healon™, a rooster-comb derived hyaluronic acid,˜4×10⁶ MW); Genzyme Corporation, Cambridge, Mass. (Surgicoat™, arecombinant hyaluronic acid); Pronova Biopolymer, Inc. Portsmouth, N.H.(Hyaluronic Acid FCH, a high molecular weight (e.g., ˜1.5-2.2×10⁶ MW)hyaluronic acid prepared from cultures of Streptococcus zooepidemicus;Sodium Hyaluronate MV, ˜1.0-1.6×10⁶ MW and Sodium Hyaluronate LV,˜1.5-2.2×10⁶ MW); Calbiochem-Novabiochem AB, Lautelfingen, Switzerland(Hyaluronic Acid, sodium salt (1997 company catalog number 385908)prepared from Streptococcus sp.); Intergen Company, Purchase, N.Y. (arooster-comb derived hyaluronic acid, >1×10⁶ MW); Diosynth Inc.,Chicago, Ill.; Amerchol Corp., Edison, N.J. and Kyowa Hakko Kogyo Co.,Ltd., Tokyo, Japan.

[0139] Once the pharmaceutical composition has been formulated, it maybe stored in sterile vials as a solution, suspension, gel, emulsion,solid, or a dehydrated or lyophilized powder. Such compositions each maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration. In a specificembodiment, the present invention is directed to kits for producing asingle-dose administration unit. The kits may each contain both a firstcontainer having a dried protein and a second container having anaqueous formulation. Kits included within the scope of this inventionare single and multi-chambered pre-filled syringes; exemplary pre-filledsyringes (e.g., liquid syringes, and lyosyringes such as Lyo-Ject®, adual-chamber pre-filled lyosyringe) are available from Vetter GmbH,Ravensburg, Germany.

[0140] Uses

[0141] TNFbp product(s) may be useful as research reagents and astherapeutic and diagnostic agents. Thus the TNFbp product(s) may be usedin in vitro and/or in vivo diagnostic assays to quantify the amount ofnative TNFR-I, sTNFR-I, TNFR-II or sTNFR-II in a tissue or organ sampleor to determine and/or isolate cells which express TNF (Scallon et al.(1995), supra). In assays of tissues or organs there will be lessradioactivity from an ¹²⁵I-TNFbp product(s) binding to TNF, as comparedto a standardized binding curve of an ¹²⁵I-TNFbp product(s), due tounlabeled native sTNFR-I or sTNFR-II binding to TNF. Similarly, the useof an ¹²⁵I-TNFbp product(s) may be used to detect the presence of TNF invarious cell types.

[0142] This invention also contemplates the use of TNFbp product(s) inthe generation of antibodies and the resultant antibodies (specificallyincluding those which also bind to native sTNFR-I or sTNFR-II).Antibodies can be developed which bind to TNFbp product(s). One ofordinary skill in the art can use well-known published procedures toobtain monoclonal, polyclonal antibodies or recombinant antibodies whichspecifically recognize and bind to the various proteins encoded by theamino acid sequences of the present invention. Such antibodies may thenbe used to purify and characterize the native sTNFR-I and nativesTNFR-II, or to quantify the number of TNFR-I or TNFR-II expressed on acell surface.

[0143] The present invention also relates to methods for the treatmentof certain diseases and medical conditions (many of which can becharacterized as inflammatory diseases) that are mediated by TNF, aswell as the related sequela and symptoms associated therewith. Anon-exclusive list of acute and chronic TNF-mediated diseases includesbut is not limited to the following: cachexia/anorexia; cancer (e.g.,leukemias); chronic fatigue syndrome; depression; diabetes (e.g.,juvenile onset Type 1 and diabetes mellitus); fibromyelgia or analgesia;graft versus host rejection; hyperalgesia; inflammatory bowel disease;ischemic, including cerebral ischemia (brain injury as a result oftrauma, epilepsy, hemorrhage or stroke, each of which may lead toneurodegeneration); lung diseases (e.g., adult respiratory distresssyndrome and pulmonary fibrosis); multiple sclerosis; neuroinflammatorydiseases; ocular diseases; pain; pancreatitis; pulmonary fibrosis;reperfusion injury; rheumatic diseases (e.g., rheumatoid arthritis,osteoarthritis, juvenile (rheumatoid) arthritis, seronegativepolyarthritis, ankylosing spondylitis, Reiter's syndrome and reactivearthritis, psoriatic arthritis, enteropathic arthritis, polymyositis,dermatomyositis, scleroderma, systemic sclerosis, vasculitis, cerebralvasculitis, Lyme disease, staphylococcal-induced (“septic”) arthritis,Sjögren's syndrome, rheumatic fever, polychondritis and polymyalgiarheumatica and giant cell arteritis); septic shock; side effects fromradiation therapy; systemic lupus erythematous; temporal mandibularjoint disease; thyroiditis; tissue transplantation or an inflammatorycondition resulting from strain, sprain, cartilage damage, trauma,orthopedic surgery, infection or other disease process.

[0144] The TNFbp product(s) may be administered to a patient intherapeutically effective amounts for the prevention or treatment ofTNF-mediated diseases, including rheumatic diseases. The term “patient”is intended to encompass animals (e.g., cats, dogs and horses) as wellas humans.

[0145] TNFbp product(s) may be administered via topical, enteral orparenteral administration including, without limitation, infusion,intraarterial, intraarticular, intracapsular, intracardiac, intradermal,intramuscular, intraorbital, intrathecal, intravenous, intraperitoneal,intraspinal, intrasternal injection, intraventricular, subcutaneous,subcuticular, subcapsular, subarachnoid and transtracheal. TNFbpproduct(s) may also be administered via oral administration or beadministered through mucus membranes, that is, buccally, intranasally,rectally or sublingually for systemic delivery.

[0146] It is preferred that TNFbp product(s) be administered viaintraarticular, intramuscular, intravenous or subcutaneous injection.Additionally, TNFbp product(s) may be administered by continuousinfusion (e.g., constant or intermittent implanted or external infusionflow-modulating devices) so as to continuously provide the desired levelof TNFbp product(s) in the blood for the duration of the administration.This is may be accomplished by means of a mini-pump, such as an osmoticmini-pump. In these ways, one can be assured that the amount of drug ismaintained at the desired level and one can take blood samples andmonitor the amount of drug in the bloodstream. Various pumps arecommercially available, for example, from suppliers such as MiniMedInc., Sylmar, Calif. (e.g., MT507) and Alza Corp., Palo Alto, Calif.(e.g. Alzet osmotic pump, model 2MLI).

[0147] It is also contemplated that other modes of continuous ornear-continuous dosing may be practiced. For example, chemicalderivatization may result in sustained release forms of the proteinwhich have the effect of continuous presence in the blood stream, inpredictable amounts based on a determined dosage regimen.

[0148] Modes of using TNFbp product(s) for the treatment of TNF-mediateddiseases, including rheumatic diseases (e.g., osteoarthritis, psoriaticarthritis and rheumatoid arthritis), are set forth in European PatentApplication 567566, the teachings of which are hereby incorporated byreference. By way of example but not limitation, in one specificembodiment, TNFbp product(s) may be administered intra-articularly forthe treatment of rheumatoid arthritis and osteoarthritis. By way ofexample but not limitation in another specific embodiment, TNFbpproduct(s) may be administered subcutaneously or intramuscularly for thetreatment of rheumatoid arthritis, inflammatory bowel disease,cachexia/anorexia or multiple sclerosis. By way of example but notlimitation, in a still further specific embodiment TNFbp product(s) maybe administered intravenously for the treatment of brain injury as aresult of trauma, epilepsy, hemorrhage or stroke; or administeredintraventricularly for the treatment of brain injury as a result oftrauma. A specific mode for the treatment of arthritis includes: (1) asingle intraarticular injection of a TNFbp product(s) given periodicallyas needed to prevent or remedy the flare-up of arthritis and (2)periodic subcutaneous injections of TNFbp product(s). In anotherspecific embodiment, a TNFbp product(s) may be administered in thetreatment of septic shock. The initiation of treatment for septic shockshould begin as soon as possible after septicemia or the chance ofsepticemia is diagnosed. For example, treatment may be begun immediatelyfollowing surgery or an accident or any other event that may carry therisk of initiating septic shock. Preferred modes for the treatment ofadult respiratory distress syndrome include: (1) single or multipleintratracheal administrations of a TNFbp product(s) and (2) bolus orcontinuous intravenous infusion of a TNFbp product(s).

[0149] In another embodiment, cell therapy is also contemplated, e.g.,implantation of cells producing a TNFbp product(s). This embodiment ofthe present invention may include implanting into patients cells whichare capable of synthesizing and secreting a TNFbp product(s). Such cellsproducing a TNFbp product(s) may be cells which do not normally producea TNFbp product(s) but which have been modified to produce a TNFbpproduct(s). The cells also may be cells whose ability to produce a TNFbpproduct(s) have been augmented by transformation with a polynucleotidesuitable for the expression and secretion of a TNFbp product(s). Inorder to minimize a potential immunological reaction in patients byadministering a TNFbp product(s) of a foreign species, it is preferredthat the cells be of the same species as the patient (e.g., human) orthat the cells be encapsulated with material that provides a barrieragainst immune recognition, or that cells be placed into animmunologically privileged anatomical location, such as in the testis,eye or central nervous system.

[0150] Human or non-human animal cells may be implanted into patients inbiocompatible, semi-permeable polymeric enclosures or membranes to allowrelease of a TNFbp product(s), but to prevent destruction of the cellsby the patient's immune system or by other detrimental factors from thesurrounding tissue. Alternatively, the patient's own cells, transformedex vivo to produce a TNFbp product(s), may be implanted directly intothe patient without such encapsulation. The methodology for the membraneencapsulation of living cells is familiar to those of ordinary skill inthe art, and the preparation of the encapsulated cells and theirimplantation in patients may be accomplished.

[0151] In yet another embodiment, in vivo gene therapy is alsoenvisioned, wherein a nucleic acid sequence encoding a TNFbp product(s)is introduced directly into a patient. For example, a nucleic acidsequence encoding a TNFbp product(s) is introduced into target cells vialocal injection of a nucleic acid construct, with or without anappropriate delivery vector, such as an adeno-associated virus vector.Alternative viral vectors include but are not limited to retrovirus,adenovirus, herpes simplex virus and papilloma virus vectors. Physicaltransfer may be achieved in vivo by local injection of the desirednucleic acid construct or other appropriate delivery vector containingthe desired nucleic acid sequence, liposome-mediated transfer, directinjection (naked DNA), receptor-mediated transfer (ligand-DNA complex)or microparticle bombardment (gene gun).

[0152] Exemplary cell and gene therapy techniques are disclosed in U.S.Pat. No. 4,892,538; U.S. Pat. No. 5,011,472; U.S. Pat. No. 5,106,627; DE4219626, WO 94/20517 and 96/22793, the disclosures of which are herebyincorporated by reference.

[0153] Regardless of the manner of administration, the treatment of aTNF-mediated disease requires a dose or total dose regimen of a TNFbpproduct(s) effective to reduce or alleviate symptoms of the disease.Factors in determining the appropriate dosage or total dose regimen caninclude the disease or condition to be treated or prevented, theseverity of the disease, the route of administration, and the age, sexand medical condition of the patient.

[0154] Further refinement of the calculations necessary to determine theappropriate dosage for treatment is routinely made by those skilled inthe art, especially in light of the dosage information and assaysdisclosed herein. The frequency of dosing also depends on thepharmacokinetic parameters of the TNFbp product(s) in the formulationused. The TNFbp product(s) may be administered once, or in cases ofsevere and prolonged disorders, administered daily in less frequentdoses or administered with an initial bolus dose followed by acontinuous dose or sustained delivery. It is also contemplated thatother modes of continuous or near-continuous dosing may be practiced.For example, chemical derivatization may result in sustained releaseforms which have the effect of a continuous presence in the bloodstream,in predictable amounts based on a determined dosage or total dosageregimen. The dosage or total dose regimen can also be determined throughthe use of known assays for determining dosages used in conjunction withappropriate dose-response data.

[0155] When administered parenterally, each unit dose, for example, maybe up to 10 mg, generally up to 15 mg and more generally up to 20 mg.When administered into an articular cavity, the pharmaceuticalcomposition is preferably administered as a single injection from, forexample, a 3 to 10 ml syringe containing a dose, for example, of betweenabout 5 mg/ml to 10 mg/ml TNFbp product(s) dissolved in isotonicphosphate buffered saline. The preparation may be administered into anarticular cavity at a frequency, for example, of once every 7 to 10days. In such a manner, the administration is continuously conducted,for example, 4 to 5 times while varying the dose if necessary.

[0156] As contemplated by the present invention, a TNFbp product(s) maybe administered as an adjunct to other therapy and also with otherpharmaceutical formulations suitable for the indication being treated. ATNFbp product(s) and any of one or more additional therapies orpharmaceutical formulations may be administered separately or incombination.

[0157] In a specific embodiment, the present invention is directed tothe use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with one or more additional TNFinhibitors for the treatment of TNF-mediated diseases, including acuteand chronic inflammation. TNF inhibitors include compounds and proteinswhich block in vivo synthesis or extracellular release of TNF, includingthe following compounds.

[0158] Additional TNF inhibitors include anti-TNF antibodies (e.g., MAK195F Fab antibody (Holler et al. (1993), 1st International Symposium onCytokines in Bone Marrow Transplantation, 147; CDP 571 anti-TNFmonoclonal antibody (Rankin et al. (1995), British Journal ofRheumatology, 34:334-342, the disclosure of which is incorporated byreference); BAY X 1351 murine anti-tumor necrosis factor monoclonalantibody (Kieft et al. (1995), 7th European Congress of ClinicalMicrobiology and Infectious Diseases, 9, the disclosure of which isincorporated by reference); CenTNF cA2 anti-TNF monoclonal antibody(Elliott et al. (1994), Lancet, 344:1125-1127 and Elliott et al. (1994),Lancet, 344:1105-1110, the disclosures of which are incorporated byreference).

[0159] In a specific embodiment, the present invention is directed tothe use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with secreted or soluble humanfas antigen or recombinant versions thereof (WO 96/20206 and Mountz etal., J. Immunology, 155:4829-4837; and EP 510 691, the disclosures ofwhich are hereby incorporated by reference). WO 96/20206 disclosessecreted human fas antigen (native and recombinant, including an Igfusion protein), methods for isolating the genes responsible for codingthe soluble recombinant human fas antigen, methods for cloning the genein suitable vectors and cell types, and methods for expressing the geneto produce the inhibitors. EP 510 691 teaches DNAs coding for human fasantigen, including soluble fas antigen, vectors expressing for said DNAsand transformants transfected with the vector. When administeredparenterally, doses of a secreted or soluble fas antigen fusion proteineach are generally from about 1 micrograms/kg to about 100micrograms/kg.

[0160] In a specific embodiment, the present invention is directed tothe use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or moreinterleukin-1 inhibitors for the treatment of TNF-mediated diseases,including acute and chronic inflammation such as cachexia/anorexia;chronic fatigue syndrome, depression; diabetes (e.g., juvenile onsetType 1 and diabetes mellitus); fibromyelgia or analgesia; graft versushost rejection; hyperalgesia, inflammatory bowel disease; ischemicinjury, including cerebral ischemia (e.g., brain injury as a result oftrauma, epilepsy, hemorrhage or stroke, each of which may lead toneurodegeneration); lung diseases (e.g., ARDS and pulmonary fibrosis);multiple sclerosis, ocular diseases; pain; pancreatitis, reperfusioninjury; rheumatic diseases (e.g., rheumatoid arthritis, osteoarthritis,juvenile (rheumatoid) arthritis, seronegative polyarthritis, ankylosingspondylitis, Reiter's syndrome and reactive arthritis, psoriaticarthritis, enteropathic arthritis, polymyositis, dermatomyositis,scleroderma, systemic sclerosis, vasculitis, cerebral vasculitis, Lymedisease, staphylococcal-induced (“septic”) arthritis, Sjögren'ssyndrome, rheumatic fever, polychondritis and polymyalgia rheumatica andgiant cell arteritis); septic shock; side effects from radiationtherapy; temporal mandibular joint disease; tumor metastasis; or aninflammatory condition resulting from strain, sprain, cartilage damage,trauma, orthopedic surgery, infection or other disease processes.Classes of interleukin-1 inhibitors include interleukin-1 receptorantagonists (any compound capable of specifically preventing activationof cellular receptors to IL-1) such as IL-1ra, as described below;anti-IL-1 receptor monoclonal antibodies (e.g., EP 623674), thedisclosure of which is hereby incorporated by reference; IL-1 bindingproteins such as soluble IL-1 receptors (e.g., U.S. Pat. No. 5,492,888,U.S. Pat. No. 5,488,032, and U.S. Pat. No. 5,464,937, U.S. Pat. No.5,319,071, and U.S. Pat. No. 5,180,812, the disclosures of which arehereby incorporated by reference); anti-IL-1 monoclonal antibodies(e.g., WO 9501997, WO 9402627, WO 9006371, U.S. Pat. No. 4,935,343, EP364778, EP 267611 and EP 220063, the disclosures of which are herebyincorporated by reference); IL-1 receptor accessory proteins (e.g., WO96/23067, the disclosure of which is hereby incorporated by reference),and other compounds and proteins which block in vivo synthesis orextracellular release of IL-1.

[0161] Interleukin-1 receptor antagonist (IL-1ra) is a human proteinthat acts as a natural inhibitor of interleukin-1. Preferred receptorantagonists, as well as methods of making and methods of using thereof,are described in U.S. Pat. No. 5,075,222; WO 91/08285; WO 91/17184; AU9173636; WO 92/16221; WO93/21946; WO 94/06457; WO 94/21275; FR 2706772;WO 94/21235; DE 4219626; WO 94/20517; WO 96/22793 and WO 97/28828 thedisclosures of which are incorporated herein by reference. The proteinsinclude glycosylated as well as non-glycosylated IL-1 receptorantagonists.

[0162] Specifically, three preferred forms of IL-1ra (IL-1raα, IL-1raβand IL-1rax), each being derived from the same DNA coding sequence, aredisclosed and described in U.S. Pat. No. 5,075,222. Methods forproducing IL-1 inhibitors, particularly IL-1ras, are also disclosed inthe U.S. Pat. No. 5,075,222. In a specific embodiment, an IL-1racontains an N-terminal methionyl group as a consequence of expression inE. coli. The present invention also includes modified IL-1ras. Themodified IL-1ras include, for example, muteins of such inhibitors inwhich a cysteine residue is substituted for an amino acid at one or moresites in the amino acid sequence of a naturally-occurring inhibitor.Such muteins may then be site-selectively reacted with functionalizedpolyethylene glycol (PEG) units or other sulfhydryl-containingpolyethers to create IL-1ra PEG species. WO 92/16221 discloses a numberof modified IL-1ra species and methods of making such PEG modifiedinhibitors.

[0163] An additional class of interleukin-1 inhibitors includescompounds capable of specifically preventing activation of cellularreceptors to IL-1. Such compounds include IL-1 binding proteins, such assoluble receptors and monoclonal antibodies. Such compounds also includemonoclonal antibodies to the receptors.

[0164] A further class of interleukin-1 inhibitors includes compoundsand proteins which block in vivo synthesis and/or extracellular releaseof IL-1. Such compounds include agents which affect transcription ofIL-1 genes or processing of IL-1 preproteins.

[0165] Present treatment of TNF-mediated diseases, including acute andchronic inflammation such as rheumatic diseases includes the use offirst line drugs for control of pain and inflammation classified asnon-steroidal, anti-inflammatory drugs (NSAIDs). Secondary treatmentsinclude corticosteroids, slow acting antirheumatic drugs (SAARDs) ordisease modifying (DM) drugs. Information regarding the followingcompounds can be found in The Merck Manual of Diagnosis and Therapy,Sixteenth Edition, Merck, Sharp & Dohme Research Laboratories, Merck &Co., Rahway, N.J. (1992) and in Pharmaprojects, PJB Publications Ltd.

[0166] In a specific embodiment, the present invention is directed tothe use of a TNFbp product(s) and any of one or more NSAIDs for thetreatment of TNF-mediated diseases, including acute and chronicinflammation such as rheumatic diseases and graft versus host disease.NSAIDs owe their anti-inflammatory action, at least in part, to theinhibition of prostaglandin synthesis (Goodman and Gilman in “ThePharmacological Basis of Therapeutics,” MacMillan 7th Edition (1985)).NSAIDs can be characterized into nine groups: (1) salicylic acidderivatives; (2) propionic acid derivatives; (3) acetic acidderivatives; (4) fenamic acid derivatives; (5) carboxylic acidderivatives; (6) butyric acid derivatives; (7) oxicams; (8) pyrazolesand (9) pyrazolones.

[0167] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or moresalicylic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. Such salicylic acid derivatives, prodrugesters and pharmaceutically acceptable salts thereof comprise:acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate diflusinal,etersalate, fendosal, gentisic acid, glycol salicylate, imidazolesalicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate,1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate,phenyl salicylate, salacetamide, salicylamide O-acetic acid, salsalateand sulfasalazine. Structurally related salicylic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group.

[0168] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or morepropionic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The propionic acid derivatives, prodrug estersand pharmaceutically acceptable salts thereof comprise: alminoprofen,benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen,flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen,ibuprofen aluminum, ibuproxam, indoprofen, isoprofen, ketoprofen,loxoprofen, miroprofen, naproxen, oxaprozin, piketoprofen, pimeprofen,pirprofen, pranoprofen, protizinic acid, pyridoxiprofen, suprofen,tiaprofenic acid and tioxaprofen. Structurally related propionic acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

[0169] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more aceticacid derivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The acetic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: acemetacin,alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin,diclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fenclozicacid, fentiazac, furofenac, glucametacin, ibufenac, indomethacin,isofezolac, isoxepac, lonazolac, metiazinic acid, oxametacin, oxpinac,pimetacin, proglumetacin, sulindac, talmetacin, tiaramide, tiopinac,tolmetin, zidometacin and zomepirac. Structurally related acetic acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

[0170] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more fenamicacid derivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The fenamic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: enfenamic acid,etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamatesodium, medofenamic acid, mefanamic acid, niflumic acid, talniflumate,terofenamate, tolfenamic acid and ufenamate. Structurally relatedfenamic acid derivatives having similar analgesic and anti-inflammatoryproperties are also intended to be encompassed by this group.

[0171] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or morecarboxylic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The carboxylic acid derivatives, prodrugesters and pharmaceutically acceptable salts thereof which can be usedcomprise: clidanac, diflunisal, flufenisal, inoridine, ketorolac andtinoridine. Structurally related carboxylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

[0172] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more butyricacid derivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The butyric acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: bumadizon,butibufen, fenbufen and xenbucin. Structurally related butyric acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

[0173] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more oxicams,prodrug esters or pharmaceutically acceptable salts thereof. Theoxicams, prodrug esters and pharmaceutically acceptable salts thereofcomprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicamand 4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4-(N-phenyl)-carboxamide.Structurally related oxicams having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

[0174] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or morepyrazoles, prodrug esters or pharmaceutically acceptable salts thereof.The pyrazoles, prodrug esters and pharmaceutically acceptable saltsthereof which may be used comprise: difenamizole and epirizole.Structurally related pyrazoles having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

[0175] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or morepyrazolones, prodrug esters or pharmaceutically acceptable saltsthereof. The pyrazolones, prodrug esters and pharmaceutically acceptablesalts thereof which may be used comprise: apazone, azapropazone,benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone,phenylbutazone, pipebuzone, propylphenazone, ramifenazone, suxibuzoneand thiazolinobutazone. Structurally related pyrazalones having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

[0176] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more of thefollowing NSAIDs: ε-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, antrafenine,bendazac, bendazac lysinate, benzydamine, beprozin, broperamole,bucolome, bufezolac, ciproquazone, cloximate, dazidamine, deboxamet,detomidine, difenpiramide, difenpyramide, difisalamine, ditazol,emorfazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole,flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene,isonixirn, lefetamine HCl, leflunomide, lofemizole, lotifazole, lysinclonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein,orpanoxin, oxaceprolm, oxapadol, paranyline, perisoxal, perisoxalcitrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone,proxazole, thielavin B, tiflamizole, timegadine, tolectin, tolpadol,tryptamid and those designated by company code number such as 480156S,AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C,CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182,KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102,PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281,SY6001, TA60, TAI-901 (4-benzoyl-1-indancarboxylic acid), TVX2706,U60257, UR2301 and WY41770. Structurally related NSAIDs having similaranalgesic and anti-inflammatory properties to the NSAIDs are alsointended to be encompassed by this group.

[0177] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or morecorticosteroids, prodrug esters or pharmaceutically acceptable saltsthereof for the treatment of TNF-mediated diseases, including acute andchronic inflammation such as rheumatic diseases, graft versus hostdisease and multiple sclerosis. Corticosteroids, prodrug esters andpharmaceutically acceptable salts thereof include hydrocortisone andcompounds which are derived from hydrocortisone, such as21-acetoxypregnenolone, alclomerasone, algestone, amcinonide,beclomethasone, betamethasone, betamethasone valerate, budesonide,chloroprednisone, clobetasol, clobetasol propionate, clobetasone,clobetasone butyrate, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacon, desonide, desoximerasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flumethasone pivalate,flunisolide, flucinolone acetonide, fluocinonide, fluorocinoloneacetonide, fluocortin butyl, fluocortolone, fluorocortolone hexanoate,diflucortolone valerate, fluorometholone, fluperolone acetate,fluprednidene acetate, fluprednisolone, flurandenolide, formocortal,halcinonide, halometasone, halopredone acetate, hydrocortamate,hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate,hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednicolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

[0178] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or moreslow-acting antirheumatic drugs (SAARDs) or disease modifyingantirheumatic drugs (DMARDS), prodrug esters or pharmaceuticallyacceptable salts thereof for the treatment of TNF-mediated diseases,including acute and chronic inflammation such as rheumatic diseases,graft versus host and multiple sclerosis. SAARDs or DMARDS, prodrugesters and pharmaceutically acceptable salts thereof comprise:allocupreide sodium, auranofin, aurothioglucose, aurothioglycanide,azathioprine, brequinar sodium, bucillamine, calcium3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine,clobuzarit, cuproxoline, cyclophosphamide, cyclosporin, dapsone,15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquinegold salt, gold sodium thiomalate, gold sodium thiosulfate),hydroxychloroquine, hydroxyurea, kebuzone, levamisole, lobenzarit,melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolatemofetil, myoral, nitrogen mustard, D-penicillamine, pyridinol imidazolessuch as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin andvincristine. Structurally related SAARDs or DMARDs having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

[0179] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more COX2inhibitors, prodrug esters or pharmaceutically acceptable salts thereoffor the treatment of TNF-mediated diseases, including acute and chronicinflammation. Examples of COX2 inhibitors, prodrug esters orpharmaceutically acceptable salts thereof include, for example,celecoxib. Structurally related COX2 inhibitors having similar analgesicand anti-inflammatory properties are also intended to be encompassed bythis group.

[0180] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or moreantimicrobials, prodrug esters or pharmaceutically acceptable saltsthereof for the treatment of TNF-mediated diseases, including acute andchronic inflammation. Antimicrobials include, for example, ampicillin,amoxycillin, aureomicin, bacitracin, ceftazidime, ceftriaxone,cefotaxime, cephachlor, cephalexin, cephradine, ciprofloxacin,clavulanic acid, cloxacillin, dicloxacillan, erythromycin,flucloxacillan, gentamicin, gramicidin, methicillan, neomycin,oxacillan, penicillin and vancomycin. Structurally relatedantimicrobials having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

[0181] In a more specific embodiment, the present invention is directedto the use of a TNFbp product(s) in combination (pretreatment,post-treatment or concurrent treatment) with any of one or more of thefollowing compounds for the treatment of TNF-mediated diseases,including acute and chronic inflammation: granulocyte colony stimulatingfactor; thalidomide; BN 50730; tenidap; E 5531; tiapafant PCA 4248;nimesulide; panavir; rolipram; RP 73401; peptide T; MDL 201,449A;(1R,3S)-Cis-1-[9-(2,6-diaminopurinyl)]-3-hydroxy-4-cyclopentenehydrochloride;(1R,3R)-trans-1-[9-(2,6-diamino)purine]-3-acetoxycyclopentane;(1R,3R)-trans-1-[9-adenyl)-3-azidocyclopentane hydrochloride and(1R,3R)-trans-1-[6-hydroxy-purin-9-yl)-3-azidocyclopentane.

[0182] It is especially advantageous to formulate compositions of theadditional anti-inflammatory compounds in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein refers to physically discrete units suited as unitary dosages forthe patients to be treated, each unit containing a predeterminedquantity of additional anti-inflammatory compounds calculated to producethe desired therapeutic effect in association with the requiredpharmaceutical carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coating,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like which are compatible with the active ingredient andwith the mode of administration and other ingredients of the formulationand not deleterious to the recipient.

[0183] For oral therapeutic administration, the additionalanti-inflammatory compound may be incorporated with excipients and usedin the form of ingestible tablets, buccal tablets, troches, capsules,elixers, suspensions, syrups, wafers and the like, or it may beincorporated directly with the food in the diet. The tablets, troches,pills, capsules and the like may also contain the following: a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,alginic acid and the like; a lubricant such as magnesium stearate; asweetening agent such as sucrose, lactose or saccharin; or a flavoringagent such as peppermint, oil of wintergreen or cherry or orangeflavoring. When the dosage unit form is a capsule, it may contain, inaddition to material of the type described herein, a liquid carrier.Various other materials may be present as a coating or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills or capsules may be coated with shellac, sugar or both. Of course,any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the additional anti-inflammatory compound may beincorporated into a sustained-release preparation and formulation. Theamount of the additional anti-inflammatory compound in suchtherapeutically useful composition is such that a suitable dosage willbe obtained.

[0184] For parenteral therapeutic administration, each additionalanti-inflammatory compound may be incorporated with a sterile injectablesolution. The sterile injectable solution may be prepared byincorporating the additional anti-inflammatory compound in the requiredamount in an appropriate pharmaceutically acceptable carrier, withvarious other ingredients, followed by filtered sterilization. In thecase of dispersions, each may be prepared by incorporating theadditional anti-inflammatory compound into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated herein. In the case of sterile injectablesolutions, each may be prepared by incorporating a powder of theadditional anti-inflammatory compound and, optionally, any additionaldesired ingredient from a previously sterile-filtered solution thereof,wherein the powder is prepared by any suitable technique (e.g., vacuumdrying and freeze drying).

[0185] The use of such media and agents is well known in the art (seefor example, Remington's Pharmaceutical Sciences, 18th Ed. (1990), MackPublishing Co., Easton, Pa. 18042, pages 1435-1712, the disclosure ofwhich is hereby incorporated by reference). Supplementary activeingredients can also be incorporated into the compositions.

[0186] The specific dose of the additional anti-inflammatory compound iscalculated according to the approximate body weight or surface area ofthe patient. Other factors in determining the appropriate dosage caninclude the acute and chronic inflammatory disease or condition to betreated or prevented, the severity of the disease, the route ofadministration and the age, sex and medical condition of the patient.Further refinement of the calculations necessary to determine theappropriate dosage for treatment involving each of the herein-mentionedformulations is routinely made by those skilled in the art. Dosages canalso be determined through the use of known assays for determiningdosages used in conjunction with appropriate dose-response data.

[0187] Thus, for example, it is within the scope of the invention thatdoses of the additional anti-inflammatory compounds selected fortreating a particular acute or chronic inflammatory disease such asrheumatic diseases can be varied to achieve a desired therapeuticeffect. Where one of the additional anti-inflammatory compounds has sideeffects, it can be given to patients during alternate treatment periodsof combination therapy. For example, chronic methotrexate treatment isassociated with gastrointestinal, hepatic, bone marrow and pulmonarytoxicity (Sandoval et al. (1995), British Journal of Rheumatology,34:49-56, the disclosure of which is hereby incorporated by reference).

[0188] Tests for monitoring the improvement of a disease can includespecific tests directed, for example, to the determination of systemicresponse to inflammation, which include the erythrocyte sedimentationrate (ESR) and acute phase reactants (APR). Observations are made of theswelling, etc. of the afflicted body parts. Improvement in stiffness,and grip (where applicable), and reduction in pain of the patient isalso observed. If the patient's condition is stable, the patient isre-treated at the same dosage weekly and is evaluated weekly. Providedthe patient's condition is stable, the treatment may be continued. Aftersix months of treatment, anatomical changes of the skeleton aredetermined by radiologic imaging, for example by X-radiography.

[0189] At the end of each period, the patient is again evaluated.Comparison of the pre-treatment and post-treatment radiologicalassessment, ESR and APR indicates the efficacy of the treatments.According to the efficacy of the treatments and the patient's condition,the dosage may be increased or maintained constant for the duration oftreatment.

[0190] Preferably, the present invention is directed to a method with,optionally, one of the following combinations to treat or preventTNF-mediated diseases, including acute and chronic inflammation such asrheumatic diseases and the symptoms associated therewith. Onecombination is a TNFbp product(s) (e.g., sTNFR-I, sTNFR-II, sTNFRfragments (2.6D sTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s)(sTNFR-I/IgG1 or sTNFR-II/IgG1) thereof) with one or more ofmethotrexate, leflunomide, an immunosuppressant (e.g., cyclosporin),ciprofloxacin, secreted or soluble fas antigen and an IL-1 inhibitor(e.g., IL-1ra). Preferred combinations include the TNFbp product(s) andmethotrexate, or the TNFbp product(s) and leflunomide. Anothercombination is a TNFbp product(s) (e.g., sTNFR-I, sTNFR-II, sTNFRfragments (2.6D sTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s)(sTNFR-I/IgG1 or sTNFR-II/IgG1) thereof) with one or more ofmethotrexate, leflunomide, sulphasazine and hydroxychloroquine.

[0191] In a specific preferred embodiment, the method comprises theadministration (e.g., intraarticular, subcutaneous or intramuscular) ofTNFbp product(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6D sTNFRssuch as 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 or sTNFR-II/IgG1),optionally formulated with a controlled release polymer (e.g., a dextranor hyaluronan)) in combination (pretreatment, post-treatment orconcurrent treatment) with methotrexate and/or leflunomide and/or anIL-1 inhibitor (e.g., IL-1ra) and/or a secreted or soluble Fas antigento treat rheumatic diseases.

[0192] In a specific preferred embodiment, the method comprises theadministration (e.g., intravenous or intraventricular) of a TNFbpproduct(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6D sTNFRs suchas 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 or sTNFR-II/IgG1),optionally formulated with a controlled release polymer (e.g., a dextranor hyaluronan)) in combination (pretreatment, post-treatment orconcurrent treatment) with tissue plasminogen activator and/or an IL-1inhibitor (e.g. IL-1ra) to treat brain injury as a result of trauma,epilepsy, hemorrhage or stroke, each of which may lead toneurodegeneration.

[0193] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous or intramuscular) of a TNFbpproduct(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6D sTNFRs suchas 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 or sTNFR-II/IgG1),optionally formulated with a controlled release polymer (e.g., a dextranor hyaluronan)) in combination (pretreatment, post-treatment orconcurrent treatment) with one or more of a corticosteroid, cyclosporin,FK-506, or an interferon (e.g., alpha interferon, beta interferon, gammainterferon or consensus interferon) and/or an IL-1 inhibitor (e.g.IL-1ra, optionally formulated with a controlled release polymer (e.g., adextran or hyaluronan)) to treat multiple sclerosis.

[0194] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous or intramuscular) of a TNFbpproduct(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6D sTNFRs suchas 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 or sTNFR-II/IgG1),optionally formulated with a controlled release polymer (e.g., a dextranor hyaluronan)) in combination (pretreatment, post-treatment orconcurrent treatment) with G-CSF and/or an IL-1 inhibitor (e.g. IL-1ra)to treat inflammatory bowel disease.

[0195] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous or intramuscular) of a TNFbpproduct(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6D sTNFRs suchas 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 or sTNFR-II/IgG1),optionally formulated with a controlled release polymer (e.g., a dextranor hyaluronan)) in combination (pretreatment, post-treatment orconcurrent treatment) with leptin, Marinol™ or Megace™ to treatcachexia/anorexia.

[0196] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous or intramuscular) of a TNFbpproduct(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6D sTNFRs suchas 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 or sTNFR-II/IgG1),optionally formulated with a controlled release polymer (e.g., a dextranor hyaluronan)) in combination (pretreatment, post-treatment orconcurrent treatment) with leptin to treat diabetes.

[0197] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous, intraventricular or intrathecal) ofa TNFbp product(s) (e.g., sTNFR-I, sTWFR-II, sTNFR fragments (2.6DsTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 orsTNFR-II/IgG1), optionally formulated with a controlled release polymer(e.g., a dextran or hyaluronan)) in combination (pretreatment,post-treatment or concurrent treatment) with an NSAID (e.g.,indomethacin) and/or an IL-1 inhibitor (e.g. IL-1ra) to treatAlzheimer's disease.

[0198] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous, intraventricular or intrathecal) ofa TNFbp product(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6DsTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 orsTNFR-II/IgG1), optionally formulated with a controlled release polymer(e.g., a dextran or hyaluronan)) optionally in combination(pretreatment, post-treatment or concurrent treatment) with a secretedor soluble fas antigen to treat cancer (e.g., leukemias); diabetes(e.g., juvenile onset Type 1 diabetes mellitus); graft versus hostrejection; hepatitis; ischemic/reperfusion injury, including cerebralischemia (brain injury as a result of trauma, epilepsy, hemorrhage orstroke, each of which may lead to neurodegeneration); neuroinflammatorydiseases; rheumatic diseases, and tissue transplantation.

[0199] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous, intraventricular or intrathecal) ofa TNFbp product(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6DsTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 orsTNFR-II/IgG1), optionally formulated with a controlled release polymer(e.g., a dextran or hyaluronan)) optionally in combination(pretreatment, post-treatment or concurrent treatment) withosteoprotogerin (European Patent Application No. 96309363.8) in thetreatment of osteoporosis or Paget's disease.

[0200] In a specific preferred embodiment, the method comprises theadministration (e.g., subcutaneous, intraventricular or intrathecal) ofa TNFbp product(s) ((e.g., sTNFR-I, sTNFR-II, sTNFR fragments (2.6DsTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 orsTNFR-II/IgG1), optionally formulated with a controlled release polymer(e.g., a dextran or hyaluronan)) in combination with gene therapy (e.g.,using the human adenovirus) to modulate 15 the inflammatory response tovector antigens (zhang et al. (1997), Arthritis & Rheumatism, 40(9):S220(1138)).

[0201] The surprising and unexpected result disclosed herein is theability of TNFbp product(s) (e.g., sTNFR-I, sTNFR-II, sTNFR fragments(2.6D sTNFRs such as 2.6D sTNFR-I) or sTNFR Fc(s) (sTNFR-I/IgG1 orsTNFR-II/IgG1), optionally formulated with a controlled release polymer(e.g., a dextran or hyaluronan)) and methotrexate to act synergisticallyin the treatment of various symptoms associated with TNF-mediateddiseases, including acute and chronic inflammation such as rheumaticdiseases. “Synergistically” is used herein to refer to a situation wherethe benefit conveyed by the joint administration of inhibitors isgreater than the algebraic sum of the effects resulting from theseparate administration of components of the combination. As shown inthe experiments below, in the adjuvant arthritis model the combinationtreatment of TNFbp product(s) and methotrexate is synergistic withrespect to treating systemic inflammation (i.e., splenomegaly) andweight loss associated with rheumatoid arthritis. Thus, the combinedtreatment with TNFbp product(s) and methotrexate has the advantage ofachieving the same result with a lower dose or less frequentadministration of methotrexate, thereby reducing any toxic effect andpotentially the advantage of persisting even after the treatment hasterminated.

[0202] Methotrexate is an anti-metabolite and immunosuppressive drug.Methotrexate is an effective anti-inflammatory agent with utility in thetreatment of severe and disabling psoriasis and rheumatoid arthritis(Hoffmeister (1983), The American Journal of Medicine, 30:69-73 andJaffe (1988), Arthritis and Rheumatism, 31:299). Methotrexate isN-[4-[(2,4-diamino-6-pteridinyl)methylamino]benzoyl]-L-glutamic acid andhas the structural formula:

[0203] The following references describe the preparation of methotrexate(Seeger et al. (1949), J. Am. Chem. Soc., 71:1753; the metabolism ofmethotrexate (Freeman (1958), J. Pharmacol. Exp. Ther, 122:154 andHenderson et al. (1965), Cancer Res., 25:1008); the toxicity ofmethotrexate (Condit et al. (1960), Cancer, 13:222-249; thepharmacokinetic models of methotrexate (Bischoff et al. (1970), J.Pharm, Sci., 59:149); the metabolism and pharmacokinetics ofmethotrexate (Evans (1980), Appl. Pharmacokinet., Williams et al.(eds.), pp. 518-548 (Appl. Ther., Inc.); the clinical pharmacology ofmethotrexate (Bertino (1981), Cancer Chemother., 3:359-375 and Jolivetet al. (1983), N. Eng. J. Med., 309:1094-1104); and the clinicalexperience of methotrexate in rheumatoid arthritis (Weinblatt et al.(1985), N. Eng. J. Med., 312:818-822; Furst (1985), J. Rheumatol.,12(12):1-14; Williams et al. (1985), Arthritis Rheum., 28:721-730 andSeitz et al. (1995), British Journal of Rheumatology, 34:602-609).Additionally, numerous patents have been issued disclosing active agentmethotrexate and methods for synthesizing methotrexate or potentialintermediates in the synthesis of methotrexate: U.S. Pat. Nos.2,512,572, 3,892,801, 3,989,703, 4,057,548, 4,067,867, 4,079,056,4,080,325, 4,136,101, 4,224,446, 4,306,064, 4,374,987, 4,421,913 and4,767,859.

[0204] The mechanism of action of methotrexate is poorly understood,however various activities of this drug have been demonstrated whichlikely contribute to its efficacy (Segal et al. (1990), Seminars inArthritis and Rheumatism, 20:190-198). The following mechanisms ofaction for methotrexate have been postulated: inhibition offolate-dependent pathways and protein metabolism (Morgan et al. (1987),Arthritis and Rheumatism, 30:1348-1356); inhibition of neutrophilmigration into arthritic joints (Van de Kerkhof et al. (1985), BritishJournal of Dermatology, 113:251-255; Ternowitz et al. (1987), Journal ofInvestigative Dermatology, 89:192-196 and Sperling (1992), Arthritis andRheumatism, 35:376-384); IL-6 inhibitory activity (Segal (1991),Arthritis and Rheumatism, 34(2):146-152) and the local specificanti-proliferative effect on cells involved in arthritis (Rodenhuis etal. (1987), Arthritis and Rheumatism, 30:369-374). Methotrexate has beenshown to block the interleukin-1 beta/interleukin-1 receptor pathway(Brody et al. (1993), European Journal of Clinical Chemistry andClinical Biochemistry, 31(10):667-674); however, although methotrexatemay inhibit the proliferative effects of IL-1 and decrease monocyte IL-1production in the short term in certain patients, this effect is notsustained and is unlikely to explain the long-term efficacy ofmethotrexate (Barrera et al. (1996), Seminars in Arthritis andRheumatism, 25(4):234-253).

[0205] Methotrexate may be administered orally, intraperitoneally,subcutaneously or intravenously. Oral administration is preferred. Thefollowing is an example of the procedure for the combined administrationof a TNFbp product(s) and methotrexate to treat a human patient. Thepatient takes a tablet or capsule of methotrexate three times a week, ata total weekly dose of 5 to 50 mg/patient/week. The patient also isinjected intravenously with TNFbp product(s), at a daily dose of 50 to150 mg. It will be appreciated by those skilled in the art that thedoses presented herein are the preferred doses. The starting dose of theparticular compound(s) used is reduced for a patient who exhibitsadverse reaction, or the drug used in combination with the compound(s)can be changed or reduced, e.g., depending on the differentformulations, routes, dose schedules and/or other variables known tothose skilled in the art, such as the individual patient's tolerance ofthe drug, its efficacy and toxicity.

[0206] Preferably, the patient is treated with a weekly starting dose ofmethotrexate at between 5 mg and 7.5 mg (orally or intramuscularly) anda daily dose of TNFbp product(s) at between 50 mg and 150 mgintravenously. The dosage of methotrexate is increased by 5 mg every 2to 3 weeks. The maximum dosage level is determined at a point at whichthe patient shows improvements, which is generally preferably less thanabout 25 mg of methotrexate per week, more preferably between 5 to 25 mgof methotrexate per week. At the end of the five-day period the patientis evaluated. The evaluation includes physical examination and extensivelaboratory testing. The tests include evaluation for toxicity.Additional laboratory monitoring in the case of methotrexate preferablyincludes a complete blood cell count every 2 weeks for the first 3months and then monthly thereafter. Additional precautions preferablyinclude monthly assessments of levels of serum albumin, alanine aminotransferase, bilirubin, creatinine and blood urea nitrogen. Monthlyurinalysis is also preferred.

[0207] The above is by way of example and does not preclude thetreatment of other inflammatory joint diseases arising from abnormal orundesirably normal immune responses. The example also does not precludeother treatments to be used concurrently with these anti-inflammatorycompounds that are known by those skilled in the art or that could bearrived at by those skilled in the art using the guidelines set forth inthis specification. Other anti-inflammatory compounds mentioned abovecan be used in combination with the treatments.

EXAMPLES

[0208] Standard methods for many of the procedures described in thefollowing examples, or suitable alternative procedures, are provided inwidely recognized manuals of molecular biology such as, for example,Sambrook et al., Molecular Cloning, Second Edition, Cold Spring HarborLaboratory Press (1987) and Ausabel et al., Current Protocols inMolecular Biology, Greene Publishing Associates/Wiley Interscience, NewYork (1990). All chemicals were either analytical grade or USP grade.

Example 1

[0209] An animal model of rheumatoid arthritis induced by an adjuvantwas used to investigate the combination therapy of a TNF binding proteinand methotrexate in male Lewis rats (3-7/group) weighing at least 200g.

[0210] On day—0, all rats were injected with 100 μl of Freunds CompleteAdjuvant (Sigma Chemical Co., St. Louis, Mo.) to which a syntheticadjuvant, N,N-dioctyldecyldecyl-N′,N-bis(2-hydroxy-ethyl)propanediamine,50 mg/ml, was added. On days 0-14 methotrexate in 1%carboxymethylcellulose (Sigma) was orally administered daily (0.06mg/kg) to two groups of rats. On days 8, 10, 12, and 14, E. coli-derivedc105 sTNFR-I dimerized with PEG-20,000-bis-vinyl sulfone (c105 sTNFR-Idumbbell; prepared generally in accordance with the teachings of WO95/34326) formulated in pharmaceutical composition (34 mM NaCl, 10 mMsodium phosphate, 4% sorbitol (w/v) in water; pH 6.5) was administeredby subcutaneous (SC) injection (3 mg/kg) to one group of rats beingtreated with both Freunds Complete Adjuvant and methotrexate and toanother group of rats being treated with Freunds Complete Adjuvantalone.

[0211] Body weights were taken on day 0 and every other day from day 9to termination on day 15. Caliper measurements and clinical scoring weredone on day 9 and every other day until termination. At this timeanimal's body, paw and spleen weights were determined.

[0212] As seen in FIGS. 3 and 4, rats treated with c105 sTNFR-I dumbbellalone exhibited about 42% inhibition of paw swelling (area under thecurve—AUC), no significant benefit on splenomegaly (not shown) and about13.2% inhibition of body weight change (not shown). Rats treated withmethotrexate had 26% inhibition of paw swelling (AUC), no inhibition ofspleen weight (not shown) and 3% inhibition of body weight change (notshown). The combination therapy provided 75% inhibition of paw swelling(AUC), 48% inhibition of splenomegaly (not shown) and 16.2% inhibitionof body weight change (not shown).

[0213] As seen in FIG. 5, the final analysis (inhibition at termination)of terminal paw weights and spleen weights indicated that c105 sTNFR-Idumbbell alone resulted in 10.9% inhibition of paw inflammation, 30.4%inhibition of splenomegaly and 13.2% inhibition of body weight change(not shown). Methotrexate treatment alone gave only a 3.9% inhibition ofpaw inflammation, 8.5% inhibition of splenomegaly and 3% inhibition ofbody weight change (not shown). The combination of c105 sTNFR-I dumbbelland methotrexate resulted in a 46.8% inhibition of paw swelling, 48%inhibition of splenomegaly and 16.2% inhibition of body weight change(not shown).

Example 2

[0214] An animal model of rheumatoid arthritis induced by an adjuvantwas used to investigate the combination therapy of a TNF binding proteinand methotrexate in male Lewis rats (5-7/group) weighing at least 200g.

[0215] On day—0, all rats were injected with 100 μl of Freunds CompleteAdjuvant (Sigma Chemical Co., St. Louis, Mo.) to which a syntheticadjuvant, N,N-dioctyldecyldecyl-N′,N-bis(2-hydroxy-ethyl)propanediamine,50 mg/ml, was added. On day 0-14 methotrexate in 1%carboxymethylcellulose (Sigma) was orally administered daily (0.06mg/kg) to two groups of rats. On days 9, 11, and 13, CHO-derivedsTNFR-II/hIgG1 fusion protein (sTNFR-II Fc; prepared generally inaccordance with the teachings of EP 418 014) formulated inpharmaceutical composition (34 mM NaCl, 10 mM sodium phosphate, 4%sorbitol (w/v) in water; pH 6.5) was administered by subcutaneousinfusion (18 mg/kg) to one group of rats being treated with both FreundsComplete Adjuvant and methotrexate and to another group of rats beingtreated with Freunds Complete Adjuvant alone.

[0216] Body weights were taken on day 0 and every other day from day 9to termination on day 15. Caliper measurements and clinical scoring weredone daily from day 9 until termination on day 15. At this time animal'sbody, paw and spleen weights were determined.

[0217] As seen in FIG. 6, rats treated with sTNFR-II Fc alone exhibitedabout 8% inhibition of paw swelling (area under the curve—AUC), with nosignificant benefit on splenomegaly (−7%) or body weight change (−5%).Rats treated with methotrexate had 66% inhibition of paw swelling (AUC),74% inhibition of spleen weight and 64% inhibition of body weightchange. The combination therapy provided 96% inhibition of paw swelling(AUC), 94% inhibition of splenomegaly and 79% inhibition of body weightchange.

[0218] As seen in FIG. 7, the final analysis (inhibition at termination)of terminal paw weights indicated that sTNFR-II Fc alone resulted in 10%inhibition of paw inflammation, methotrexate treatment alone gave a 74%inhibition of paw inflammation and the combination of sTNFR-II Fc andmethotrexate resulted in a 88% inhibition of paw swelling.

Example 3

[0219] The combination immunotherapeutic effects of c105 sTNFR-Idumbbell and fas fusion protein were assessed using a mouse model ofD-Galactosamine (D-GalNH₂) induced lethality. The D-galactosamine(D-GalNH2)/Lipopolysaccharide (LPS) model (Mountz et al., J. Immunology,155:4829-4837). In this model, MRL-1pr/1pr autoimmune mice areadministered D-GalNH₂with bacterial endotoxin (LPS), and lethality isobserved through +96 hours post challenge.

[0220] Materials and Methods:

[0221] Dihydrofolate reductase (DHFR) deficient Chinese hamster ovarycells (CHOd-cells) were transfected with fas/hIgG1 chimeric cDNA(Mountz, et al. (1996), “Autoimmunity Due to Defective Nur-77, Fas andTNF-R1 Apoptosis” in Mechanisms of Lymphocyte Activation and ImmuneRegulation, Vol. 6, p241-262 (Gupta and Cohen (Eds)), Plenem Press,N.Y.) in pDSRα2, generally in accordance with the disclosure ofDeClerck, et al. (1991), JCB, 266:3893-3899. The transfection procedurediffered from the protocol of set forth in DeClerck, et al. (1991),supra, as follows: the cells were transfected with 800,000 cells, with10 micrograms and 8 micrograms of herring sperm as a carrier, and thecells were split at 2 days post-transfection.

[0222] Following expression of the fas fusion protein, the protein waspurified using a Protein G Sepharose Fast Flow, generally in accordancewith Jungbauer, et al. (1989), J. Chrom., 476:257-268. The purifiedprotein was formulated in Phosphate buffered saline (Gibco BRL, GrandIsland, N.Y.).

[0223] Protocol:

[0224] After overnight fasting, 6-8 week old female MRL-1pr/1pr mice(Jackson Laboratory, Bar Harbor, Me.) (5/7/group) were cannulated withjugular catheters and allowed to recover for several days. They werethen placed in infusion cages and acclimated for a week prior initiatingsaline infusion.

[0225] At hour—0, all mice were injected intraperitoneally with 31micrograms of D-GalNH₂ (Sigma) suspended in Hank's Balanced SaltSolution (Gibco BRL) (120 micrograms/ml); and lipopolysaccharide (LPS)from E. coli Serotype 0127:B8 (Sigma) in sterile, endotoxin-freephosphate buffered saline (PBS) (6 micrograms/mouse).

[0226] At 0—hour±2 hours post-challenge, fas fusion protein formulatedin a pharmaceutical composition (Phosphate buffered saline (Gibco BRL,Grand Island, N.Y.)) was administered intravenously in serial 2-folddilutions (microgram/kg dosages) to two groups of mice.

[0227] At 0—hour-±2 hours post-challenge, c105 sTNFR-I dumbbellformulated in a pharmaceutical composition (34 mM NaCl, 10 mM sodiumphosphate, 4% sorbitol (w/v) in water; pH 6.5) was administeredintravenously in serial 2-fold dilutions (microgram/kg dosages) to onegroup of mice being treated with both D GalNH₂ and fas fusion proteinand to another group of mice being treated with D-GalNH₂ alone.

[0228] ED₅₀ curves were generated with statistical software for theMacIntosh (Statview®, Mountain View, Calif.). Lethality was followedthrough +96 hour after challenge.

[0229] Results:

[0230] As seen in FIG. 8, mice administered c105 sTNFR-I dumbbell (100micrograms/kg; N=6; I.V.) at time=−1 hour before challenge were observedto be completely protected (100% survival) against LPS challenge incomparison to control (saline-treated) mice (N=6) challenged withLPS/D-GalNH₂ (P<0.01). Mice treated with sub-optimal doses of c105sTNFR-I dumbbell (25 micrograms/kg; N=6) were observed to have ˜35%protection through +96 hours after challenge. All mice treated with fasfusion protein (100 micrograms/kg; N=6) were dead by +24 hours-postchallenge. However, when mice (N=6) were treated I.V. with both c105sTNFR-I dumbbell (25 micrograms/kg) and fas fusion protein (100micrograms/kg), enhanced survival (70%) was observed through +36 hoursin comparison to either the c105 sTNFR-I dumbbell treated (25micrograms), fas fusion protein (100 micrograms/kg), or disease controlanimals alone (P<0.05). These results suggest that c105 sTNFR-I dumbbelland fas fusion protein are synergistic in their therapeutic effects inthe LPS/D-GalNH_(2.) model of acute inflammation.

[0231] The foregoing description of the invention is exemplary forpurposes of illustration and explanation. It will be apparent to thoseskilled in the art that changes and modifications are possible withoutdeparting from the spirit and scope of the invention. It is intendedthat the following claims be interpreted to embrace all such changes andmodifications.

1 4 483 base pairs nucleic acid unknown unknown cDNA CDS 1..483 1 GATAGT GTG TGT CCC CAA GGA AAA TAT ATC CAC CCT CAA AAT AAT TCG 48 Asp SerVal Cys Pro Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser 1 5 10 15 ATTTGC TGT ACC AAG TGC CAC AAA GGA ACC TAC TTG TAC AAT GAC TGT 96 Ile CysCys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys 20 25 30 CCA GGCCCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC 144 Pro Gly ProGly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser 35 40 45 TTC ACC GCTTCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA 192 Phe Thr Ala SerGlu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys 50 55 60 TGC CGA AAG GAAATG GGT CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC 240 Cys Arg Lys Glu MetGly Gln Val Glu Ile Ser Ser Cys Thr Val Asp 65 70 75 80 CGG GAC ACC GTGTGT GGC TGC AGG AAG AAC CAG TAC CGG CAT TAT TGG 288 Arg Asp Thr Val CysGly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp 85 90 95 AGT GAA AAC CTT TTCCAG TGC TTC AAT TGC AGC CTC TGC CTC AAT GGG 336 Ser Glu Asn Leu Phe GlnCys Phe Asn Cys Ser Leu Cys Leu Asn Gly 100 105 110 ACC GTG CAC CTC TCCTGC CAG GAG AAA CAG AAC ACC GTG TGC ACC TGC 384 Thr Val His Leu Ser CysGln Glu Lys Gln Asn Thr Val Cys Thr Cys 115 120 125 CAT GCA GGT TTC TTTCTA AGA GAA AAC GAG TGT GTC TCC TGT AGT AAC 432 His Ala Gly Phe Phe LeuArg Glu Asn Glu Cys Val Ser Cys Ser Asn 130 135 140 TGT AAG AAA AGC CTGGAG TGC ACG AAG TTG TGC CTA CCC CAG ATT GAG 480 Cys Lys Lys Ser Leu GluCys Thr Lys Leu Cys Leu Pro Gln Ile Glu 145 150 155 160 AAT 483 Asn 161amino acids amino acid linear protein 2 Asp Ser Val Cys Pro Gln Gly LysTyr Ile His Pro Gln Asn Asn Ser 1 5 10 15 Ile Cys Cys Thr Lys Cys HisLys Gly Thr Tyr Leu Tyr Asn Asp Cys 20 25 30 Pro Gly Pro Gly Gln Asp ThrAsp Cys Arg Glu Cys Glu Ser Gly Ser 35 40 45 Phe Thr Ala Ser Glu Asn HisLeu Arg His Cys Leu Ser Cys Ser Lys 50 55 60 Cys Arg Lys Glu Met Gly GlnVal Glu Ile Ser Ser Cys Thr Val Asp 65 70 75 80 Arg Asp Thr Val Cys GlyCys Arg Lys Asn Gln Tyr Arg His Tyr Trp 85 90 95 Ser Glu Asn Leu Phe GlnCys Phe Asn Cys Ser Leu Cys Leu Asn Gly 100 105 110 Thr Val His Leu SerCys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys 115 120 125 His Ala Gly PhePhe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn 130 135 140 Cys Lys LysSer Leu Glu Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu 145 150 155 160 Asn705 base pairs nucleic acid unknown unknown cDNA CDS 1..705 3 TTG CCCGCC CAG GTG GCA TTT ACA CCC TAC GCC CCG GAG CCC GGG AGC 48 Leu Pro AlaGln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 ACA TGCCGG CTC AGA GAA TAC TAT GAC CAG ACA GCT CAG ATG TGC TGC 96 Thr Cys ArgLeu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 AGC AAG TGCTCG CCG GGC CAA CAT GCA AAA GTC TTC TGT ACC AAG ACC 144 Ser Lys Cys SerPro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 TCG GAC ACC GTGTGT GAC TCC TGT GAG GAC AGC ACA TAC ACC CAG CTC 192 Ser Asp Thr Val CysAsp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 TGG AAC TGG GTT CCCGAG TGC TTG AGC TGT GGC TCC CGC TGT AGC TCT 240 Trp Asn Trp Val Pro GluCys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 GAC CAG GTG GAA ACTCAA GCC TGC ACT CGG GAA CAG AAC CGC ATC TGC 288 Asp Gln Val Glu Thr GlnAla Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 ACC TGC AGG CCC GGC TGGTAC TGC GCG CTG AGC AAG CAG GAG GGG TGC 336 Thr Cys Arg Pro Gly Trp TyrCys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 CGG CTG TGC GCG CCG CTGCGC AAG TGC CGC CCG GGC TTC GGC GTG GCC 384 Arg Leu Cys Ala Pro Leu ArgLys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 AGA CCA GGA ACT GAA ACATCA GAC GTG GTG TGC AAG CCC TGT GCC CCG 432 Arg Pro Gly Thr Glu Thr SerAsp Val Val Cys Lys Pro Cys Ala Pro 130 135 140 GGG ACG TTC TCC AAC ACGACT TCA TCC ACG GAT ATT TGC AGG CCC CAC 480 Gly Thr Phe Ser Asn Thr ThrSer Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 CAG ATC TGT AAC GTGGTG GCC ATC CCT GGG AAT GCA AGC AGG GAT GCA 528 Gln Ile Cys Asn Val ValAla Ile Pro Gly Asn Ala Ser Arg Asp Ala 165 170 175 GTC TGC ACG TCC ACGTCC CCC ACC CGG AGT ATG GCC CCA GGG GCA GTA 576 Val Cys Thr Ser Thr SerPro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190 CAC TTA CCC CAG CCAGTG TCC ACA CGA TCC CAA CAC ACG CAG CCA ACT 624 His Leu Pro Gln Pro ValSer Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205 CCA GAA CCC AGC ACTGCT CCA AGC ACC TCC TTC CTG CTC CCA ATG GGC 672 Pro Glu Pro Ser Thr AlaPro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 CCC AGC CCC CCA GCTGAA GGG AGC ACT GGC GAC 705 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp225 230 235 235 amino acids amino acid linear protein 4 Leu Pro Ala GlnVal Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys ArgLeu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys CysSer Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp ThrVal Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp Asn TrpVal Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp GlnVal Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr CysArg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 ArgLeu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Arg AspAla 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro GlyAla Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His ThrGln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe LeuLeu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp225 230 235

We claim:
 1. A method for treating an acute or chronic inflammatorydisease which comprises administering to a patient in need thereoftherapeutically effective amounts of a TNF binding protein and at leastone additional anti-inflammatory drug, wherein said TNF binding proteinand additional anti-inflammatory drug are administered separately or incombination.
 2. The method of claim 1 wherein the anti-inflammatory drugis methotrexate(N-[4-[[2,4-diamino-6-pteridinyl)methylamino]benzoyl]-L-glutamic acid).3. The method of claim 1 wherein the anti-inflammatory drug is a fasfusion protein.
 4. The method of claim 1, wherein said TNF bindingprotein is wherein said TNF binding protein is sTNFR-I, sTNFR-II, sTNFRfragments or sTNFR Fc.
 5. The method of any one of claims 1 through 4,wherein said inflammatory disease is an inflammatory disease of a joint.6. The method of claim 5, wherein said inflammatory disease of a jointis rheumatoid arthritis.
 7. The method of claim 3, wherein said TNFbinding protein and said methotrexate are administered in apharmaceutically acceptable carrier.
 8. The method of claim 3, whereinsaid TNF binding protein and said fas fusion protein are administered ina pharmaceutically acceptable carrier.
 9. A pharmaceutical compositioncomprising a TNF binding protein and an additional anti-inflammatorydrug.
 10. The pharmaceutical composition wherein the anti-inflammatorydrug is methotrexate.
 11. The pharmaceutical composition wherein theanti-inflammatory drug is a fas fusion protein.
 12. The pharmaceuticalcomposition of claim 9, wherein said TNF binding protein is sTNFR-I,sTNFR-II, sTNFR fragments or sTNFR Fc.
 13. The pharmaceuticalcomposition of claim 9, wherein said TNF binding protein is present inan amount of up to about 20 mg.
 14. The pharmaceutical composition ofclaim 10, wherein said methotrexate is present in an amount of up toabout 25 mg.
 15. A use of an anti-inflammatory drug, other than anon-TNF binding protein, in the preparation of a medicament for treatingan acute or chronic inflammatory disease in a mammal in combination withthe administration of a TNF binding protein.
 16. The use of claim 15,wherein the anti-inflammatory drug is methotrexate.
 17. The useaccording to claim 16 wherein the methotrexate in the medicament is upto about 25 mg.
 18. The use according to claims 15 through 17 whereinsaid methotrexate is administered orally, intraperitoneally,subcutaneously or intravenously.
 19. The use according to claims 15through 17 wherein said methotrexate is administered orally.
 20. The useof claim 15, wherein the anti-inflammatory drug is a fas fusion protein.21. A use of a TNF binding protein in the preparation of a medicamentfor treating an acute or chronic inflammatory disease in a mammal incombination with the administration of an additional anti-inflammatorydrug.
 22. The use of according to claim 21, wherein theanti-inflammatory drug is methotrexate.
 23. The use according to claims20 through 22 wherein said methotrexate is administered orally,intraperitoneally, subcutaneously or intravenously.
 24. The use ofaccording to claim 21, wherein the anti-inflammatory drug is a fasfusion protein.
 25. The use according to claims 21 through 24 whereinthe TNF binding protein is sTNFR-I, sTNFR-II, sTNFR fragments or sTNFRFc.
 26. The use according to claims 21 through 25 wherein the TNFbinding protein in the medicament is present in an amount of up to about200 mg.